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https://f1000research.com/articles/5-1467/v1
|
22 Jun 16
|
{
"type": "Review",
"title": "Stroke rehabilitation research needs to be different to make a difference",
"authors": [
"Cathy M. Stinear"
],
"abstract": "Stroke continues to be a major cause of adult disability. In contrast to progress in stroke prevention and acute medical management, there have been no major breakthroughs in rehabilitation therapies. Most stroke rehabilitation trials are conducted with patients at the chronic stage of recovery and this limits their translation to clinical practice. Encouragingly, several multi-centre rehabilitation trials, conducted during the first few weeks after stroke, have recently been reported; however, all were negative. There is a renewed focus on improving the quality of stroke rehabilitation research through greater harmonisation and standardisation of terminology, trial design, measures, and reporting. However, there is also a need for more pragmatic trials to test interventions in a way that assists their translation to clinical practice. Novel interventions with a strong mechanistic rationale need to be tested in both explanatory and pragmatic trials if we are to make a meaningful difference to stroke rehabilitation practice and outcomes.",
"keywords": [
"Stroke",
"rehabilitation",
"intervention assessment"
],
"content": "Background\n\nStroke is a leading cause of adult disability worldwide1. Advances in stroke prevention have led to a decline in stroke incidence, particularly in developed countries2. There have also been recent advances in acute stroke treatment with thrombolysis and clot retrieval3. However, the number of people living with the consequences of stroke continues to rise2. Stroke rehabilitation has steadily evolved with new service delivery models and a greater understanding of the importance of therapy intensity and task specificity. However, the search continues for new therapies that can be widely incorporated into routine clinical practice, despite more than 1000 randomised controlled trials (RCTs) having been conducted4.\n\nOne factor that may limit the translational impact of stroke rehabilitation RCTs is their timing. It is important to conduct rehabilitation trials during the initial days and weeks after stroke because this is when spontaneous biological recovery (SBR) is taking place5 and when rehabilitation is delivered in the ‘real world’. Testing an intervention at the time of its intended use is crucial for evaluating its efficacy as well as its feasibility in clinical practice. We have found that over half of motor rehabilitation RCTs are conducted with patients who are at least 6 months post-stroke, when rehabilitation services are no longer available to most patients6. Only 5% of RCTs were of good quality and recruited all patients within 30 days of stroke. Of these, half were negative. Compared with negative trials, the positive trials were more likely to recruit fewer than 40 patients and have no follow-up measures. There is clearly a need for larger trials conducted early after stroke in the real-world clinical setting.\n\nIn the last 18 months, six multi-centre rehabilitation RCTs that recruited patients within the first 3 months after stroke have been reported. These trials are summarised below.\n\n1. SIRRACT (Stroke Inpatient Rehabilitation Reinforcement of ACTivity) recruited 135 patients within 45 days after stroke at 16 sites over the course of 15 months7. Participants were randomly assigned to either standardised verbal feedback about walking speed or augmented feedback based on activity graphs derived from wireless activity sensors. The primary outcomes were average daily time spent walking during inpatient rehabilitation and the fastest safe 15-metre walking speed at discharge from inpatient rehabilitation.\n\n2. CIRCIT (Circuit class therapy or seven-day week therapy for Increasing Rehabilitation Intensity of Therapy after stroke) recruited 283 patients between 5 and 197 days (mean of 28 days) after stroke at five sites in 36 months8. Participants were randomly assigned to usual care therapy 5 days per week, usual care therapy 7 days per week, or circuit class therapy 5 days per week. The primary outcome was the 6-minute walk test at 4 weeks post-randomisation.\n\n3. AVERT (A Very Early Rehabilitation Trial) recruited 2104 patients within 24 hours of stroke symptom onset at 56 sites over the course of 100 months9. Participants were randomly assigned to usual care or very early mobilisation, which required at least three additional out-of-bed sessions targeting standing and walking beginning within 24 hours of stroke. The primary outcome was a favourable outcome 3 months after stroke, defined as a modified Rankin Scale score of 0, 1, or 2.\n\n4. A trial of acupuncture recruited 862 patients between 3 and 10 days after stroke at 40 sites in 35 months10. Participants were randomly assigned to either usual care alone or with the addition of acupuncture 5 days per week for 3 weeks. The primary outcome was death or disability at 6 months post-stroke, defined as a Barthel Index score of not more than 60 points.\n\n5. The ICARE (Interdisciplinary Comprehensive Arm Rehabilitation Evaluation) trial recruited 361 patients between 16 and 106 days (mean of 46 days) after stroke at seven sites in 45 months11. Participants were randomly assigned to usual and customary care, a 30-hour programme of task-oriented motor rehabilitation for the upper limb delivered over 10 weeks, or dose-equivalent usual and customary upper-limb therapy. The primary outcome was the change in the log-transformed time score from the Wolf Motor Function Test, between baseline and 12 months after randomisation.\n\n6. The SWIFT (Soft-Scotch Walking Initial FooT) Cast trial recruited 105 patients between 3 and 42 days (mean of 21 days) after stroke at two sites in 25 months. Participants were randomly assigned to conventional physical therapy with a conventional ankle-foot orthosis or with a customised ankle-foot orthosis12. The primary outcome was walking speed at the end of the 6-week intervention.\n\nNone of these trials found that the intervention was superior to standard care, and one found that the intervention worsened outcomes9. These are disappointing results for many of the clinicians and researchers who worked on the trials, for the funding bodies, and most importantly for the patients involved and the wider stroke community. Although these trials at least demonstrate that large multi-centre rehabilitation trials can be conducted at the subacute stage of stroke, it should also be noted that two of the larger trials took several years to complete, recruited less than 10% of screened patients, and had recruitment rates less than one patient per month per site9,11. A low proportion of patients recruited raises potential concerns about the generalisability of the intervention, whereas a slow recruitment rate raises potential concerns about the feasibility of similar studies in the future.\n\nLow and slow recruitment can be the product of strict inclusion/exclusion criteria, typical of explanatory trials designed to show that a standardised treatment is efficacious in a carefully selected group of patients. Although these are features of a well-designed study, they can also limit the trial’s usefulness to real-world clinical practice. If the treatment is found to be beneficial, the clinician does not know whether to use it for a patient who would have been excluded from the trial or in a setting that cannot provide the precisely defined treatment.\n\nThe issue of generalisability has long been recognised13 and needs to be explicitly addressed in stroke rehabilitation research. As a simple starting point, an intervention’s generalisability could be more easily appreciated if trials reported the proportion of patients for whom an intervention is suitable, even if they cannot participate in research because of factors such as reduced capacity for consent, having contraindications to research measures such as magnetic resonance imaging, or being enrolled in another study. This has been reported by two rehabilitation RCTs that recruited all patients within 30 days of stroke. One reported that the intervention was suitable for 40% of all admitted stroke patients and 9% were eligible for participation in the trial14. The other reported that the intervention was suitable for 11% of all admitted stroke patients and 3% were eligible for participation in the trial15. Distinguishing between eligibility for the intervention and eligibility for research provides a clearer picture of the proportion of patients who could potentially benefit from the intervention if it were part of routine clinical practice.\n\n\nWhat might need to change in order to achieve a breakthrough in stroke rehabilitation?\n\nTwo reports published in March addressed this important question. The first, from Juka Jolkkonen and Gert Kwakkel, made several useful recommendations16. These include standardisation of terminology for recovery, greater treatment contrast between the experimental and control arms of trials, clearer definition of ‘usual and standard care’ when it is the control condition, and the use of outcome measures that can distinguish between true neurological recovery and adaptation or compensation strategies. These authors also identify low sample size as a major problem for most stroke rehabilitation trials16, although solving this problem alone with large multi-centre trials is unhelpful if other aspects of trial design are not also improved.\n\nThe second report, from Julie Bernhardt and colleagues on behalf of the Stroke Recovery and Rehabilitation Roundtable17, clearly identifies many limitations in current stroke rehabilitation research and signals broad agreement that these must be overcome in order to advance rehabilitation research. Some of the limitations noted are that the theoretical or mechanistic basis of interventions is often not well articulated, the dose of the intervention can seem arbitrary and poorly controlled, and the timing and type of outcome measures used are highly variable between trials. The report advocates the use of more clinically relevant animal models, agreed biomarkers for patient stratification, the systematic reporting of interventions and their fidelity, and the use of a core set of outcome measures made at agreed time points.\n\nWhat about the interventions themselves? Those tested in the six studies summarised above were variations on current practice, possibly because these are easiest to evaluate within the clinical setting, using existing staff and resources. Even the large, well-designed and carefully controlled studies outlined above were unable to detect a difference between standard care and a variation of standard care in the ‘noisy’ environment of SBR. This may be unsurprising given recent evidence that current therapy practice does not seem to interact with SBR18,19. Greater contrast is needed16, as well as the evaluation of novel treatments that bear little resemblance to current therapy practice and directly target the neurobiological processes responsible for recovery5. This might require ‘aspirational’ trials20 of interventions that could not be delivered with current rehabilitation resources and service delivery models.\n\nAssuming there are interventions that can meaningfully enhance recovery during the initial weeks after stroke, how might these eventually translate to widespread clinical practice? A further consideration, not explicitly made by recent reports16,17, is that translation requires a more pragmatic trial design. Pragmatic trials are designed and reported in a way that helps clinicians evaluate how and with whom the intervention could be used in clinical practice13. They often take place in a usual care setting, with minimal patient selection, and with a level of flexibility in delivery of the treatment that would be found if it were part of usual practice13,20. However, this runs counter in some respects to the greater levels of standardisation advocated for in recent reports16,17. Highly standardised protocols are an important component of explanatory trials, which aim to detect treatment efficacy under ideal conditions that minimise the messiness of real-world clinical practice. Researchers could aim at the design stage to more consciously position their trials on the explanatory-pragmatic continuum by using tools such as PRECIS (Pragmatic-Explanatory Continuum Indicatory Summary)21. Trials at both ends of the continuum are needed and can be equally rigorous; they are simply designed to answer two different questions22. Finally, a novel rehabilitation intervention is highly unlikely to work for everyone, and it does not need to. But it is more likely to become part of clinical practice if trials are designed and reported23 in a way that allows clinicians to judge which patients are most likely to benefit from it in the real-world rehabilitation setting.\n\n\nConclusions\n\nThe neutral outcomes of several large multi-centre rehabilitation trials conducted during spontaneous recovery from stroke7–12 give pause for thought. These recent trials represent steps in a useful direction, towards testing interventions at the time when most stroke rehabilitation takes place. They seem to have also prompted discussion about how best to advance stroke rehabilitation research through improved and harmonised trial design16,17. The need for greater contrast between experimental interventions and standard care, and the potential importance of enhancing SBR, are increasingly apparent. Interventions that are efficacious during spontaneous recovery also need to be trialled with a more pragmatic approach to evaluate their effectiveness and facilitate their translation to clinical practice. Clearly, we need to be doing something different in order to make a difference.\n\n\nAbbreviations\n\nRCT, randomised controlled trial; SBR, spontaneous biological recovery.",
"appendix": "Competing interests\n\n\n\nThe author is a member of the Stroke Recovery and Rehabilitation Roundtable.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe author thanks Winston Byblow and Marie-Claire Smith for useful discussion on these topics.\n\n\nReferences\n\nMurray CJ, Barber RM, Foreman KJ, et al.: Global, regional, and national disability-adjusted life years (DALYs) for 306 diseases and injuries and healthy life expectancy (HALE) for 188 countries, 1990-2013: quantifying the epidemiological transition. Lancet. 2015; 386(10009): 2145–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFeigin VL, Forouzanfar MH, Krishnamurthi R, et al.: Global and regional burden of stroke during 1990–2010: findings from the Global Burden of Disease Study 2010. Lancet. 2014; 383(9913): 245–54. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nCampbell BC, Donnan GA, Lees KR, et al.: Endovascular stent thrombectomy: the new standard of care for large vessel ischaemic stroke. Lancet Neurol. 2015; 14(8): 846–54. PubMed Abstract | Publisher Full Text\n\nMcIntyre A, Campbell N, Vermeer J, et al.: Methodological Quality of Motor Intervention Randomized Controlled Trials in Stroke Rehabilitation. J Stroke Cerebrovasc Dis. 2016; 25(2): 248–53. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKrakauer JW, Carmichael ST, Corbett D, et al.: Getting neurorehabilitation right: what can be learned from animal models? Neurorehabil Neural Repair. 2012; 26(8): 923–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStinear C, Ackerley S, Byblow W, et al.: Rehabilitation is initiated early after stroke, but most motor rehabilitation trials are not: a systematic review. Stroke. 2013; 44(7): 2039–45. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDorsch AK, Thomas S, Xu X, et al.: SIRRACT: An International Randomized Clinical Trial of Activity Feedback During Inpatient Stroke Rehabilitation Enabled by Wireless Sensing. Neurorehabil Neural Repair. 2015; 29(5): 407–15. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nEnglish C, Bernhardt J, Crotty M, et al.: Circuit class therapy or seven-day week therapy for increasing rehabilitation intensity of therapy after stroke (CIRCIT): a randomized controlled trial. Int J Stroke. 2015; 10(4): 594–602. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBernhardt J, Langhorne P, Lindley RI, et al.: Efficacy and safety of very early mobilisation within 24 h of stroke onset (AVERT): a randomised controlled trial. Lancet. 2015; 386(9988): 46–55. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nZhang S, Wu B, Liu M, et al.: Acupuncture efficacy on ischemic stroke recovery: multicenter randomized controlled trial in China. Stroke. 2015; 46(5): 1301–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWinstein CJ, Wolf SL, Dromerick AW, et al.: Effect of a Task-Oriented Rehabilitation Program on Upper Extremity Recovery Following Motor Stroke: The ICARE Randomized Clinical Trial. JAMA. 2016; 315(6): 571–81. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nPomeroy VM, Rowe P, Clark A, et al.: A Randomized Controlled Evaluation of the Efficacy of an Ankle-Foot Cast on Walking Recovery Early After Stroke: SWIFT Cast Trial. Neurorehabil Neural Repair. 2016; 30(1): 40–8. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRothwell PM: External validity of randomised controlled trials: “to whom do the results of this trial apply?”. Lancet. 2005; 365(9453): 82–93. PubMed Abstract | Publisher Full Text\n\nStinear CM, Petoe MA, Anwar S, et al.: Bilateral priming accelerates recovery of upper limb function after stroke: a randomized controlled trial. Stroke. 2014; 45(1): 205–10. PubMed Abstract | Publisher Full Text\n\nDromerick AW, Lang CE, Birkenmeier RL, et al.: Very Early Constraint-Induced Movement during Stroke Rehabilitation (VECTORS): A single-center RCT. Neurology. 2009; 73(3): 195–201. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJolkkonen J, Kwakkel G: Translational Hurdles in Stroke Recovery Studies. Transl Stroke Res. 2016. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBernhardt J, Borschmann K, Boyd L, et al.: Moving rehabilitation research forward: Developing consensus statements for rehabilitation and recovery research. Int J Stroke. 2016; 11(4): 454–8. PubMed Abstract | Publisher Full Text\n\nByblow WD, Stinear CM, Barber PA, et al.: Proportional recovery after stroke depends on corticomotor integrity. Ann Neurol. 2015; 78(6): 848–59. PubMed Abstract | Publisher Full Text\n\nFeng W, Wang J, Chhatbar PY, et al.: Corticospinal tract lesion load: An imaging biomarker for stroke motor outcomes. Ann Neurol. 2015; 78(6): 860–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTreweek S, Zwarenstein M: Making trials matter: pragmatic and explanatory trials and the problem of applicability. Trials. 2009; 10: 37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThorpe KE, Zwarenstein M, Oxman AD, et al.: A pragmatic-explanatory continuum indicator summary (PRECIS): a tool to help trial designers. J Clin Epidemiol. 2009; 62(5): 464–75. PubMed Abstract | Publisher Full Text\n\nLurie JD, Morgan TS: Pros and cons of pragmatic clinical trials. J Comp Eff Res. 2013; 2(1): 53–8. PubMed Abstract | Publisher Full Text\n\nZwarenstein M, Treweek S, Gagnier JJ, et al.: Improving the reporting of pragmatic trials: an extension of the CONSORT statement. BMJ. 2008; 337: a2390. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "14532",
"date": "22 Jun 2016",
"name": "Julie Bernhardt",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14533",
"date": "22 Jun 2016",
"name": "Robert Teasell",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1467
|
https://f1000research.com/articles/5-1466/v1
|
22 Jun 16
|
{
"type": "Review",
"title": "Chemotherapy-induced peripheral neuropathy: an update on the current understanding",
"authors": [
"James Addington",
"Miriam Freimer",
"Miriam Freimer"
],
"abstract": "Chemotherapy-induced peripheral neuropathy is a common side effect of selected chemotherapeutic agents. Previous work has suggested that patients often under report the symptoms of chemotherapy-induced peripheral neuropathy and physicians fail to recognize the presence of such symptoms in a timely fashion. The precise pathophysiology that underlies chemotherapy-induced peripheral neuropathy, in both the acute and the chronic phase, remains complex and appears to be medication specific. Recent work has begun to demonstrate and further clarify potential pathophysiological processes that predispose and, ultimately, lead to the development of chemotherapy-induced peripheral neuropathy. There is increasing evidence that the pathway to neuropathy varies with each agent. With a clearer understanding of how these agents affect the peripheral nervous system, more targeted treatments can be developed in order to optimize treatment and prevent long-term side effects.",
"keywords": [
"peripheral neuropathy",
"Chemotherapy-induced peripheral neuropathy",
"CIPN",
"Taxanes",
"Platinum agents",
"Vinca alkaloids"
],
"content": "Introduction\n\nWith the advancement of better cancer therapies, including targeted chemotherapeutic agents, come longer patient survival times and the potential for long-term treatment-related side effects. Chemotherapy-induced peripheral neuropathy (CIPN) can be a severe side effect often associated with several chemotherapeutic agents including the platinum agents, taxanes, vinca alkaloids, thalidomide, and bortezomib. CIPN is often dose dependent and progressive while receiving and after such treatment1–3. In severe cases, the pain, sensory changes, and weakness associated with CIPN can lead to dose reductions, changes in chemotherapy protocols, or termination of a therapeutic agent. The morbidity associated with CIPN can lead to pronounced alterations in quality of life and independent performance of activities of daily living4,5. Unfortunately, the specific pathogenesis and pathophysiological effects of specific agents are poorly understood4,6. A recent meta-analysis of more than 4000 chemotherapy-treated patients found the prevalence of CIPN to be 68.1% within the first month of chemotherapy treatment, 60.0% at 3 months, and 30.0% at 6 months6. With such a high prevalence of disease, better understanding of the pathophysiology, early recognition of those at risk, and implementation of potential treatments are crucial to the prevention and management of CIPN. This review aims to focus on an update of the underlying pathophysiology leading to CIPN and will briefly discuss newer treatment trials and updates. As the purpose of this review is to update readers on more recent advancements, previously known work will be only briefly reviewed for familiarity and new theories will be explored in more detail.\n\n\nPathophysiology/diagnostics\n\nThe manner in which selected chemotherapeutic agents result in CIPN has been extensively studied for many years. Numerous studies have demonstrated treatment-specific mechanisms by which such agents affect the peripheral nervous system. Disrupted microtubule-mediated axonal transport, axonal degeneration, direct damage to the dorsal root ganglion, and mitochondrial dysfunction have all been shown in previous studies. Krøigård et al. showed that although patients treated with oxaliplatin or docetaxel may have purely sensory symptoms and signs, electrodiagnostic testing may demonstrate both motor and sensory involvement. Additionally, a cohort of patients may have sensory symptoms yet have normal nerve conduction studies. Further evaluation with skin biopsy and/or quantitative sensory testing may suggest predominately small-fiber neuropathy7. Laser Doppler imaging, a newer technique used to evaluate small-fiber neuropathies, has been shown to be more specific in terms of evaluating small-fiber dysfunction in CIPN patients8. High-resolution ultrasound is another recently introduced technology to evaluate peripheral neuropathies. In non-CIPN axonal neuropathies, the cross-sectional area of a visualized nerve on ultrasound is typically reduced in comparison to compressive neuropathies (i.e. median neuropathy at the wrist, ulnar neuropathy at the elbow). Interestingly, in patients with no history of compressive neuropathies but with axonal neuropathies by nerve conduction studies presumed secondary to chemotherapy, the cross-sectional area of visualized nerves is increased.\n\nAlthough the neuropathy is felt to be axonal in CIPN, this raises questions as to why the cross-sectional area is increased and whether these patients are more prone to compressive neuropathies9.\n\n\nPlatinum agents\n\nThe platinum agents, more specifically carboplatin and oxaliplatin, exert their anti-neoplastic effects by forming platinum-DNA adducts that ultimately lead to cell cycle arrest and apoptosis. Used for the treatment of many solid malignancies, the platinum-based agents have been associated with severe side effects, including, but not limited to, nephrotoxicity and neurotoxicity. The dorsal root ganglion is not protected by the blood-brain barrier, making the DNA within the cell body of the dorsal root ganglion preferentially susceptible to toxic agents, such as the platinum agents10,11. As a result of dorsal root ganglion damage, the neurotoxicity associated with the platinum agents presents as a sensory neuronopathy with anterograde axonal degeneration.\n\nThe platinum agent oxaliplatin is known to cause a rapid, sensory neuropathy, with some studies reporting upwards of 90% of patients being affected12. The chronic neuropathy associated with oxaliplatin is present in approximately 10% of patients two years post-administration13. Electrophysiologic studies have also shown persistent low sensory amplitudes out of proportion to motor studies in chronic versus acute oxaliplatin-associated neuropathy14.\n\nIn addition to alterations at the DNA level, the platinum agents have long been felt to exert some of their neurotoxicity through alterations in transmembrane receptors and channels. Recent work has been instrumental in further elucidating these changes. Specifically, some of the neurotoxicity associated with oxaliplatin is presumed secondary to an underlying channelopathy. Oxalate, a metabolite of oxaliplatin, is known to prolong the open phase of voltage-gated Na+ channels, leading to prolonged depolarization and nerve hyperexcitability15.\n\nAnother more recent development in terms of CIPN pathophysiology relates to the functioning of transient receptor potential (TRP) channels, which are also affected by the platinum agents. The TRP channels are non-selective cation channels activated by such things as heat, acidic environments, and capsaicin. Recent work has shown that patients treated with paclitaxel or bortezomib (discussed later) showed upregulation of the TRPV1 subtype in the dorsal root ganglion16. Furthermore, treatment of pain with TRPV1 antagonists in some rat models of CIPN has been successful17. An additional mechanism of neuropathy of the platinum agents may involve certain membrane transporters. Both copper and organic cation transporters have been shown to facilitate the transport of carboplatin into the dorsal root ganglion of sensory nerves. In cell lines overexpressing the CTR1 gene, carboplatin uptake is higher and preferentially localizes to the dorsal root ganglion, not the linked axon, supporting the clinical phenotype of a sensory neuronopathy18. Studies have also shown that the organic cation transporter (OCT) class, specifically subtypes OCT1 and OCT2, are associated with the dorsal root ganglion and that mRNA levels are affected by the administration of carboplatin19,20.\n\n\nTaxanes\n\nThe taxane class of chemotherapeutic agents exerts its anti-neoplastic effects on the microtubule during the cell cycle. Loss of depolymerization of the microtubule leads to mitotic arrest during the G2/M phase of the cell cycle21. The microtubule plays several crucial functions in maintaining the integrity and health of properly functioning axons. Patients receiving one of the taxanes often develop a length-dependent sensory neuropathy, as supported by pathological specimens demonstrating axonal degeneration and secondary demyelination in a length-dependent manner22. In addition to microtubule stabilization secondary to prevention of depolymerization, previous studies have also shown mitochondrial damage in those treated with paclitaxel23. Studies completed in 2011 showed that deficits in complexes I and II were seen in the sciatic nerves of those treated with paclitaxel. In rat model studies, treatment with acetyl-L-carnitine has been shown to improve the peripheral neuropathy, supporting the theory of mitotoxicity as a potential contributing factor24. Lastly, studies have also shown that TRP upregulation in the dorsal root ganglion leads to neuropathic pain, as mentioned previously. Increased expression of TRPV4 has been demonstrated in the dorsal root ganglion of those treated with paclitaxel. TRPV4 knockout mouse studies have demonstrated an improvement in the neuropathic pain25.\n\nMore recently, studies in zebrafish have shown that changes in the epidermis may account for some of the clinicopathological changes that occur. Paclitaxel-treated zebrafish demonstrate increases in epidermal matrix-metalloproteinase-13. Treating them with corresponding inhibitors improved their response to noxious stimuli. Similarly, fluorescein-labeled paclitaxel accumulated in the basal keratinocytes of zebrafish treated with paclitaxel, while the corresponding uptake in axons was less. This suggests that changes at the epidermal level may contribute to the underlying pathophysiology of the painful neuropathy associated with paclitaxel26. Furthermore, studies in rats have shown increases in macrophages within the dorsal root ganglion. Increases in Toll-like receptor 4 leads to increased expression of monocyte chemotactic protein 1, ultimately leading to macrophage infiltration. Administration of clodronate depleted macrophage infiltration at the level of the dorsal root ganglion, improving the rats’ pain tolerance. Similarly, administering antagonists to both Toll-like receptor 4 and monocyte chemotactic protein 1 blocked the hypersensitivity experienced by the rats. Collectively, this study suggested that administration of paclitaxel to rats leads to increased Toll-like receptor 4 expression and upregulation of monocyte chemotactic protein 1 at the level of the dorsal root ganglion. Subsequently, increased macrophage infiltration leads to the production of inflammatory mediators. Blocking anywhere along this pathway improves the hypersensitivity27.\n\nIn a recent prospective trial of patients treated with adjuvant oxaliplatin (for colon cancer) or docetaxel (for high-risk breast cancer), the incidence of significant neuropathy differed between the two groups. The incidence of cold-associated allodynia was almost non-existent in the docetaxel-treated patients in comparison to their oxaliplatin counterparts. Although neuropathic symptoms were present in both treatment groups, the severity was much worse in the oxaliplatin cohort. At one-year follow up, 63.6% of patients treated with oxaliplatin met diagnostic criteria for CIPN, while only 44.8% did so in the docetaxel group28.\n\n\nVinca alkaloids\n\nThe vinca alkaloid class, most commonly vincristine, can also lead to a painful peripheral neuropathy. Similar to the taxane class, the vinca alkaloids exert their anti-neoplastic effect on the microtubule during the cell cycle, ultimately leading to cell cycle arrest. Although this is the aim for cancer cells, undifferentiated effects at the peripheral nervous system can lead to unwanted side effects. Unlike the peripheral neuropathy seen with the taxanes, the vinca alkaloids often cause sensory and motor neuropathy29. Vinca alkaloids have a high affinity for a-tubulin of the microtubules, affecting their assembly and ultimately leading to cell death30. Recent studies in models of vincristine-induced allodynia have shown a decreased level of endomorphin-2, which is primarily found in the spinal cord and exerts its analgesic effect on mu-opioid receptors. It is proposed that such changes may lead to the hypersensitivity and allodynia experienced by patients31. Furthermore, increased serine protease, which inactivates the endomorphins, was also found in the spinal cord. Blocking the serine protease pathway with diprotin A blocked the downregulation of endomorphin-2. Other studies have shown that reactive oxygen species, often produced by chemotherapy, affect serine protease activity32. Afferent pain pathways also appear to be affected, as levels of c-Fos, a pre-synaptic marker, are unregulated at the spinal cord level. This suggests functional alterations along afferent pain pathways from the peripheral to central nervous system33. Furthermore, piccolo, a molecule crucial for the maintenance of synaptic plasticity, is increased in both intermediate (III-IV) and superficial (I-II) laminae, suggesting the possibility of enhanced hyperactivity34.\n\n\nThalidomide\n\nThalidomide was introduced for the treatment of multiple myeloma in 1999. Since that time, it has become standard of care in many protocols and has been utilized in other inflammatory conditions such as inflammatory bowel disease (i.e. Crohn’s disease). The effects of thalidomide are pleiotropic, making its specific mechanism of action leading to neurotoxicity unclear. The peripheral neuropathy associated with thalidomide treatment is often sensory or sensorimotor in nature35. This usually begins within a few months of treatment onset but may persist after cessation of the agent36. Interestingly, cases of autonomic dysfunction, manifesting as both orthostatic hypotension and symptomatic bradycardia, have also been described with thalidomide treatment37. One clear mechanism of action of thalidomide is through its anti-angiogenic properties. It has been proposed that thalidomide may affect neuronal survival secondary to microvascular changes as a result of its anti-angiogenic properties. Additionally, it has been shown to downregulate tumor necrosis factor alpha (TNFα), ultimately leading to inhibition of nuclear factor kappa beta (NFκβ). Inhibition of NFκβ has been previously shown to lead to neuronal death, another proposed mechanism for the neurotoxic effects of thalidomide and its potential role in the development of peripheral neuropathy. More recently, in 2007, lenalidomide, a structural analog to thalidomide, was studied in the management of multiple myeloma. Although lenalidomide comes with its own side effect profile, less than 10% of patients treated developed a severe, grade 3–4, neuropathy. In addition, many of the patients in the study were previously treated with thalidomide and their peripheral neuropathy did not worsen during the course of treatment with lenalidomide38.\n\n\nBortezomib\n\nBortezomib is a more recent addition to cancer chemotherapeutics approved by the FDA in 2003 for the treatment of advanced myeloma. Bortezomib exerts its mechanism of action by inhibiting the 26S ribosome subunit and preventing protein degradation, leading to cell cycle arrest and apoptosis39. In patients treated with bortezomib, a severe, painful, sensory neuropathy commonly develops40,41. Bortezomib has been shown to affect polymerization of α-tubulin and result in microtubule stabilization, similar to that of the taxane class42. Mitochondria in the dorsal root ganglia of treated patients have shown vacuolation, presumed secondary to mitochondrial enlargement, which leads to activation of pro-apoptotic pathways43. Inflammation and oxidation stress have also been implicated in the development of neuropathic pain associated with the administration of bortezomib. Inhibition of NFκB by bortezomib leads to increased TNFα and the production of reactive oxygen species, both of which have been associated with neuropathic pain44,45. Another mechanism of neuropathic pain in patients treated with bortezomib is activation of TRP channels. As mentioned earlier, increased levels of TRPV1 in the dorsal root ganglion of patients treated with bortezomib have been felt to be causative of some of the neuropathic pain appreciated by patients17.\n\n\nRisk factors\n\nThe ability to predict how patients will respond or be adversely affected by specific therapies or interventions has a meaningful impact on the utility of medical treatment. An understanding of how the pre-morbid health status may impact response and side effects of chemotherapy is essential. Several risk factors have been identified which may predispose the patient to neuropathy, and diabetes mellitus is the most important. A retrospective study of 374 patients treated with taxane-based chemotherapy regimens identified 81 individuals (21.6%) as having diabetes mellitus at the time of treatment. In those diagnosed with diabetes mellitus for more than five years, the incidence of neuropathy was 75% over the course of treatment, in comparison to 48.8% and 52.8% for non-diabetics and those diagnosed for less than five years, respectively46. Although this study suggests that upwards of half of those treated with taxane-based chemotherapy will develop a neuropathy, the percentage significantly increases in those with a longer-standing history of diabetes mellitus, irrespective of their current control status. In addition to pre-morbid conditions, genetic susceptibility has been investigated in recent years. Pharmacogenomics, or more appropriately toxicogenomics, is an area of science that attempts to describe genetic predispositions to developing specific conditions. For example, under homeostatic conditions, CEP72 is involved in the proper functioning and maintenance of microtubules. In children diagnosed with acute lymphoblastic leukemia and treated with vincristine, those with single nucleotide polymorphisms affecting the promoter region of the CEP72 gene were more likely to develop a grade 2–4 neuropathy during the course of their treatment47.\n\n\nTreatment\n\nEffective management for CIPN is lacking and therefore treatment options are limited. A 2015 publication by Majithia et al. reviewed several clinical trials sponsored by the National Cancer Institute aimed at addressing this void. Several of the more recent studies between 2014 and 2015 will now be reviewed. Alpha-lipoic acid (ALA) has been studied as a prophylactic agent for non-CIPNs, for example diabetic neuropathy. A randomized, double-blinded, placebo-controlled trial in patients exposed to platinum-based agents showed no statistically significant difference in those treated with ALA 600 mg three times daily versus placebo, although there was a high dropout rate amongst patients48. A study of patients treated with oxaliplatin-based regimens showed no significant improvement in neuropathy outcomes in those treated with calcium and/or magnesium before and after infusions49. As mentioned previously, neurotoxicity from platinum-based agents may be due to accumulation in the dorsal root ganglion. The anti-oxidant glutathione has been thought to counteract such toxicity. In a randomized trial of patients receiving carboplatin and paclitaxel, there was no significant improvement in measured outcomes in patients treated with glutathione compared to those treated with placebo50.\n\nThere have been few recent studies examining the management of established CIPN. A 2014 study evaluated the combination of topical 2% ketamine plus 4% amitriptyline in patients with established CIPN, classified as taxane induced or non-taxane induced. Similar to most other studies in CIPN, there was no statistically significant difference with regard to those treated with the topical ointment versus placebo. Interestingly, those treated in the taxane-induced group reported improved symptoms, regardless of the treatment arm51. In terms of CIPN pain, the selective serotonin-norepinephrine reuptake inhibitor duloxetine has shown promise. A 2013 double-blinded, placebo-controlled, crossover trial found that duloxetine 60 mg daily had a beneficial effect on CIPN-related pain52. These findings were more recently confirmed in a study comparing duloxetine 40 mg daily and vitamin B1253.\n\n\nConclusion\n\nIn conclusion, CIPN is a complex topic. No single unifying pathophysiologic process can be identified to explain the various neuropathies that occur after exposure to different chemotherapeutic agents. Although the anti-neoplastic features of these agents are well described, the neurotoxic side effects may be multifactorial and unrelated to the anti-neoplastic pathway. As we learn more about chemotherapeutic-specific neuropathic pathways, certain targeted medications, whether preventive or treatment related, may prove to be more appropriate depending on which chemotherapy agent a patient is exposed to. Although most CIPN is related to dysfunction of the peripheral nervous system, several studies have shown functional changes near and within the central nervous system, which may lead to radical changes in terms of optimal management. Additionally, the upsurge in genetics research, the field of pharmacogenomics, and tailoring each individual patient’s treatment plan based on their genetic predispositions is radically changing healthcare. As much of chemotherapy is tailored to genetics, it may also be crucial to consider this in consideration for potential medication side effects. While it is tempting to consider chemotherapy-induced neuropathy as a single entity, it may be more pragmatic and useful to consider each neuropathy separately as a specific side effect of a specific drug class, as successful prevention and/or treatment may differ. The paucity of beneficial treatments (and preventive measures) of CIPN makes the understanding of the underlying pathophysiological processes of CIPN an imperative on the road to the development of more targeted treatment options.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nChaudhry V, Rowinsky EK, Sartorius SE, et al.: Peripheral neuropathy from taxol and cisplatin combination chemotherapy: clinical and electrophysiological studies. Ann Neurol. 1994; 35(3): 304–11. PubMed Abstract | Publisher Full Text\n\nKautio A, Haanpää M, Kautiainen H, et al.: Burden of chemotherapy-induced neuropathy--a cross-sectional study. Support Care Cancer. 2011; 19(12): 1991–6. PubMed Abstract | Publisher Full Text\n\nSmith EM, Cohen JA, Pett MA, et al.: The reliability and validity of a modified total neuropathy score-reduced and neuropathic pain severity items when used to measure chemotherapy-induced peripheral neuropathy in patients receiving taxanes and platinums. Cancer Nurs. 2010; 33(3): 173–83. PubMed Abstract | Publisher Full Text\n\nHausheer FH, Schilsky RL, Bain S, et al.: Diagnosis, management, and evaluation of chemotherapy-induced peripheral neuropathy. Semin Oncol. 2006; 33(1): 15–49. PubMed Abstract | Publisher Full Text\n\nShimozuma K, Ohashi Y, Takeuchi A, et al.: Taxane-induced peripheral neuropathy and health-related quality of life in postoperative breast cancer patients undergoing adjuvant chemotherapy: N-SAS BC 02, a randomized clinical trial. Support Care Cancer. 2012; 20(12): 3355–64. PubMed Abstract | Publisher Full Text\n\nSeretny M, Currie GL, Sena ES, et al.: Incidence, prevalence, and predictors of chemotherapy-induced peripheral neuropathy: A systematic review and meta-analysis. Pain. 2014; 155(12): 2461–70. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKrøigård T, Schrøder HD, Qvortrup C, et al.: Characterization and diagnostic evaluation of chronic polyneuropathies induced by oxaliplatin and docetaxel comparing skin biopsy to quantitative sensory testing and nerve conduction studies. Eur J Neurol. 2014; 21(4): 623–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSharma S, Venkitaraman R, Vas PR, et al.: Assessment of chemotherapy-induced peripheral neuropathy using the LDIFLARE technique: a novel technique to detect neural small fiber dysfunction. Brain Behav. 2015; 5(7): e00354.PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBriani C, Campagnolo M, Lucchetta M, et al.: Ultrasound assessment of oxaliplatin-induced neuropathy and correlations with neurophysiologic findings. Eur J Neurol. 2013; 20(1): 188–92. 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PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLiu JJ, Jamieson SM, Subramaniam J, et al.: Neuronal expression of copper transporter 1 in rat dorsal root ganglia: association with platinum neurotoxicity. Cancer Chemother Pharmacol. 2009; 64(4): 847–56. PubMed Abstract | Publisher Full Text\n\nCavaletti G, Ceresa C, Nicolini G, et al.: Neuronal drug transporters in platinum drugs-induced peripheral neurotoxicity. Anticancer Res. 2014; 34(1): 483–6. PubMed Abstract | F1000 Recommendation\n\nYonezawa A, Inui K: Organic cation transporter OCT/SLC22A and H+/organic cation antiporter MATE/SLC47A are key molecules for nephrotoxicity of platinum agents. Biochem Pharmacol. 2011; 81(5): 563–8. PubMed Abstract | Publisher Full Text\n\nJordan MA, Wilson L: Microtubules as a target for anticancer drugs. Nat Rev Cancer. 2004; 4(4): 253–65. PubMed Abstract | Publisher Full Text\n\nSahenk Z, Barohn R, New P, et al.: Taxol neuropathy. Electrodiagnostic and sural nerve biopsy findings. Arch Neurol. 1994; 51(7): 726–9. PubMed Abstract | Publisher Full Text\n\nAndré N, Braguer D, Brasseur G, et al.: Paclitaxel induces release of cytochrome c from mitochondria isolated from human neuroblastoma cells'. Cancer Res. 2000; 60(19): 5349–53. PubMed Abstract\n\nZheng H, Xiao WH, Bennett GJ: Functional deficits in peripheral nerve mitochondria in rats with paclitaxel- and oxaliplatin-evoked painful peripheral neuropathy. Exp Neurol. 2011; 232(2): 154–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlessandri-Haber N, Dina OA, Joseph EK, et al.: Interaction of transient receptor potential vanilloid 4, integrin, and SRC tyrosine kinase in mechanical hyperalgesia. J Neurosci. 2008; 28(5): 1046–57. PubMed Abstract | Publisher Full Text\n\nLisse TS, Middleton LJ, Pellegrini AD, et al.: Paclitaxel-induced epithelial damage and ectopic MMP-13 expression promotes neurotoxicity in zebrafish. Proc Natl Acad Sci U S A. 2016; 113(15): E2189–98. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nZhang H, Li Y, de Carvalho-Barbosa M, et al.: Dorsal root ganglion infiltration by macrophages contributes to paclitaxel chemotherapy-induced peripheral neuropathy. J Pain. 2016; pii: S1526-5900(16)00562-9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nVentzel L, Jensen AB, Jensen AR, et al.: Chemotherapy-induced pain and neuropathy: a prospective study in patients treated with adjuvant oxaliplatin or docetaxel. Pain. 2016; 157(3): 560–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPostma TJ, Benard BA, Huijgens PC, et al.: Long-term effects of vincristine on the peripheral nervous system. J Neurooncol. 1993; 15(7): 23–7. PubMed Abstract | Publisher Full Text\n\nLobert S, Vulevic B, Correia JJ: Interaction of vinca alkaloids with tubulin: a comparison of vinblastine, vincristine, and vinorelbine. Biochemistry. 1996; 35(21): 6806–14. PubMed Abstract | Publisher Full Text\n\nYang Y, Zhang Y, Lin G, et al.: Spinal changes of a newly isolated neuropeptide endomorphin-2 concomitant with vincristine-induced allodynia. PLoS One. 2014; 9(2): e89583. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nWang CH, Cherng WJ, Yang NI, et al.: Cyclosporine increases ischemia-induced endothelial progenitor cell mobilization through manipulation of the CD26 system. Am J Physiol Regul Integr Comp Physiol. 2008; 294(3): R811–8. PubMed Abstract | Publisher Full Text\n\nThibault K, Rivals I, M'Dahoma S, et al.: Structural and molecular alterations of primary afferent fibres in the spinal dorsal horn in vincristine-induced neuropathy in rat. J Mol Neurosci. 2013; 51(3): 880–92. PubMed Abstract | Publisher Full Text\n\nXiao WH, Bennett GJ: Chemotherapy-evoked neuropathic pain: Abnormal spontaneous discharge in A-fiber and C-fiber primary afferent neurons and its suppression by acetyl-L-carnitine. Pain. 2008; 135(3): 262–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGiannini F, Volpi N, Rossi S, et al.: Thalidomide-induced neuropathy: a ganglionopathy? Neurology. 2003; 60(5): 877–8. PubMed Abstract | Publisher Full Text\n\nMileshkin L, Stark R, Day B, et al.: Development of neuropathy in patients with myeloma treated with thalidomide: patterns of occurrence and the role of electrophysiologic monitoring. J Clin Oncol. 2006; 24(27): 4507–14. PubMed Abstract | Publisher Full Text\n\nCoutsouvelis J, Corallo CE: Thalidomide-induced bradycardia and its management. Med J Aust. 2004; 180(7): 366–7. PubMed Abstract\n\nDimopoulos M, Spencer A, Attal M, et al.: Lenalidomide plus dexamethasone for relapsed or refractory multiple myeloma. N Engl J Med. 2007; 357(21): 2123–32. PubMed Abstract | Publisher Full Text\n\nCurran MP, McKeage K: Bortezomib: a review of its use in patients with multiple myeloma. Drugs. 2009; 69(7): 859–88. PubMed Abstract | Publisher Full Text\n\nMeregalli C, Ceresa C, Canta A, et al.: CR4056, a new analgesic I2 ligand, is highly effective against bortezomib-induced painful neuropathy in rats. J Pain Res. 2012; 5: 151–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCavaletti G, Gilardini A, Canta A, et al.: Bortezomib-induced peripheral neurotoxicity: a neurophysiological and pathological study in the rat. Exp Neurol. 2007; 204(1): 317–25. PubMed Abstract | Publisher Full Text\n\nPoruchynsky MS, Sackett DL, Robey RW, et al.: Proteasome inhibitors increase tubulin polymerization and stabilization in tissue culture cells: a possible mechanism contributing to peripheral neuropathy and cellular toxicity following proteasome inhibition. Cell Cycle. 2008; 7(7): 940–9. PubMed Abstract | Publisher Full Text\n\nLandowski TH, Megli CJ, Nullmeyer KD, et al.: Mitochondrial-mediated disregulation of Ca2+ is a critical determinant of Velcade (PS-341/bortezomib) cytotoxicity in myeloma cell lines. Cancer Res. 2005; 65(9): 3828–36. PubMed Abstract | Publisher Full Text\n\nShahshahan MA, Beckley MN, Jazirehi AR: Potential usage of proteasome inhibitor bortezomib (Velcade, PS-341) in the treatment of metastatic melanoma: basic and clinical aspects. Am J Cancer Res. 2011; 1(7): 913–24. PubMed Abstract | Free Full Text\n\nChiorazzi A, Canta A, Meregalli C, et al.: Antibody against tumor necrosis factor-α reduces bortezomib-induced allodynia in a rat model. Anticancer Res. 2013; 33(12): 5453–9. PubMed Abstract\n\nKus T, Aktas G, Kalender ME, et al.: Taxane-induced peripheral sensorial neuropathy in cancer patients is associated with duration of diabetes mellitus: a single-center retrospective study. Support Care Cancer. 2016; 24(3): 1175–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDiouf B, Crews KR, Lew G, et al.: Association of an inherited genetic variant with vincristine-related peripheral neuropathy in children with acute lymphoblastic leukemia. JAMA. 2015; 313(8): 815–23. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nGuo Y, Jones D, Palmer JL, et al.: Oral alpha-lipoic acid to prevent chemotherapy-induced peripheral neuropathy: a randomized, double-blind, placebo-controlled trial. Support Care Cancer. 2014; 22(5): 1223–31. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLoprinzi CL, Qin R, Dakhil SR, et al.: Phase III randomized, placebo-controlled, double-blind study of intravenous calcium and magnesium to prevent oxaliplatin-induced sensory neurotoxicity (N08CB/Alliance). J Clin Oncol. 2014; 32(10): 997–1005. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeal AD, Qin R, Atherton PJ, et al.: North Central Cancer Treatment Group/Alliance trial N08CA-the use of glutathione for prevention of paclitaxel/carboplatin-induced peripheral neuropathy: a phase 3 randomized, double-blind, placebo-controlled study. Cancer. 2014; 120(12): 1890–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGewandter JS, Mohile SG, Heckler CE, et al.: A phase III randomized, placebo-controlled study of topical amitriptyline and ketamine for chemotherapy-induced peripheral neuropathy (CIPN): a University of Rochester CCOP study of 462 cancer survivors. Support Care Cancer. 2014; 22(7): 1807–14. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSmith EM, Pang H, Cirrincione C, et al.: Effect of duloxetine on pain, function, and quality of life among patients with chemotherapy-induced painful peripheral neuropathy: a randomized clinical trial. JAMA. 2013; 309(13): 1359–67. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHirayama Y, Ishitani K, Sato Y, et al.: Effect of duloxetine in Japanese patients with chemotherapy-induced peripheral neuropathy: a pilot randomized trial. Int J Clin Oncol. 2015; 20(5): 866–71. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "14528",
"date": "22 Jun 2016",
"name": "Amanda Peltier",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14527",
"date": "22 Jun 2016",
"name": "Jonathan D. Glass",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1466
|
https://f1000research.com/articles/5-606/v1
|
08 Apr 16
|
{
"type": "Research Article",
"title": "Lovastatin lactone may improve irritable bowel syndrome with constipation (IBS-C) by inhibiting enzymes in the archaeal methanogenesis pathway",
"authors": [
"Steven M. Muskal",
"Joe Sliman",
"John Kokai-Kun",
"Mark Pimentel",
"Vince Wacher",
"Klaus Gottlieb",
"Joe Sliman",
"John Kokai-Kun",
"Mark Pimentel",
"Vince Wacher",
"Klaus Gottlieb"
],
"abstract": "Methane produced by the methanoarchaeon Methanobrevibacter smithii (M. smithii) has been linked to constipation, irritable bowel syndrome with constipation (IBS-C), and obesity. Lovastatin, which demonstrates a cholesterol-lowering effect by the inhibition of HMG-CoA reductase, may also have an anti-methanogenesis effect through direct inhibition of enzymes in the archaeal methanogenesis pathway. We conducted protein-ligand docking experiments to evaluate this possibility. Results are consistent with recent clinical findings.METHODS: F420-dependent methylenetetrahydromethanopterin dehydrogenase (mtd), a key methanogenesis enzyme with a known sequence but no tertiary protein structural information, was modeled for two different methanogenic archaea: M. smithii and Methanopyrus kandleri. Once protein models were developed, ligand-binding sites were identified. Multiple ligands and their respective protonation, isomeric and tautomeric representations were docked into each site, including F420-coenzyme (natural ligand), lactone and β-hydroxyacid forms of lovastatin and simvastatin, and other co-complexed ligands found in related crystal structures.RESULTS: 1) Generally, for each modeled site the lactone form of the statins had more favorable site interactions compared to F420; 2) The statin lactone forms generally had the most favorable docking scores, even relative to the native template PDB ligands; and 3) The statin β-hydroxyacid forms had less favorable docking scores, typically scoring in the middle with some of the F420 tautomeric forms. Consistent with these computational results were those from a recent phase II clinical trial (NCT02495623) with a proprietary, modified-release lovastatin-lactone (SYN-010) in patients with IBS-C, which showed a reduction in symptoms and breath methane levels, compared to placebo.CONCLUSION: The lactone form of lovastatin exhibits preferential binding over the native-F420 coenzyme ligand in silico and thus could inhibit the activity of the key M. smithii methanogenesis enzyme mtd in vivo. Statin lactones may thus exert a methane-reducing effect that is distinct from cholesterol lowering activity, which requires HMGR inhibition by statin β-hydroxyacid forms.",
"keywords": [
"IBS",
"IBS-C",
"Lovastatin",
"homology modeling",
"multi-site docking"
],
"content": "Introduction\n\nIrritable bowel syndrome (IBS) affects as many as 45 million people in the United States, and up to 23% of the worldwide population1. Depending on the region, as many as 43.3% of these patients will have irritable bowel syndrome with constipation (IBS-C)2. The illness affects both men and women; however, two-thirds of diagnosed sufferers are women. Studies have linked methane production to the pathogenesis of constipation and IBS, as well as obesity3. Methanogens – i.e. anaerobes that respire hydrogen to produce methane - are found in many habitats supporting anaerobic biodegradation of organic compounds, including human and animal intestinal tracts4,5. Archaea are the only confirmed, naturally occurring biological sources of methane. Methanobrevibacter smithii (M. smithii) is the predominant methanogen in the human intestine accounting for 94% of the methanogen population3.\n\nThe isoprenoid biosynthesis for the main cell membrane components in archaea (archaeol) relies on the same enzyme that catalyzes the biosynthesis of the isoprenoid cholesterol in humans - HMG-CoA reductase (mevalonate pathway)6. It has been previously suggested that statins, i.e. known HMG-CoA reductase inhibitors, can also interfere with the biosynthesis of the archaeal cell membrane and thus inhibit archaeal growth7. Statins, specifically lovastatin, have been shown to lower methanogenesis in human stool samples8 and can inhibit archaeal cell membrane biosynthesis without affecting bacterial numbers as demonstrated in livestock and humans. Lovastatin is a secondary metabolite produced during fungal growth and is found in oyster mushrooms9, red yeast rice10, and Pu-erh11.\n\nHumans and archaea utilize the HMGR-I isoform for isoprenoid biosynthesis12. Mevastatin and lovastatin were both shown to inhibit growth of several rumen Methanobrevibacter isolates in the ~10 nmol/ml range3. While it is believed that statins inhibit methane production via their effect on cell membrane biosynthesis mediated by inhibition of HMG-CoA reductase, there is accumulating evidence for an alternative or additional mechanism of action where statins inhibit methanogenesis directly13. In one case, in silico molecular docking of the methanogenic enzyme F420-dependent NADP oxidoreductase (fno) showed that both lovastatin and mevastatin had higher affinities for the F420 binding site on fno than did F420 itself. It has been suggested that lovastatin may act as an inhibitor of fno14.\n\nSeveral reviews have appeared describing the reduction of CO2 to CH4 in methanoarchaea15. Considering other mechanisms by which statins may inhibit methanogenesis directly, we have explored two important dehydrogenases in the main methanogenesis pathway, including F420-dependent methylenetetrahydromethanopterin dehydrogenase of M. smithii [A5UMI1- 275 amino acid residues], and evolutionarily related F420-dependent methylenetetrahydromethanopterin (methylene-H(4)MPT) dehydrogenase (mtd) of Methanopyrus kandleri [Q02394 – 358 amino acid residues]. Both only leverage F420 as a coenzyme, which assisted our computational analyses by avoiding issues associated with an NADP induced fit. The A5UMI1 sequence does not have crystallographic structural information in the Protein Data Bank (PDB)16, so we needed to identify acceptable templates to model this sequence. The Q02394 sequence, however matched the 3IQZ co-complex with methylenetetrahydromethanopterin (H4M) having 52% sequence homology. While both sequences required modeling, we needed to identify one or more acceptable templates for A5UMI1. After modeling and receptor site identification, we docked and rank-ordered multiple ligand variations across several modeled receptor sites to evaluate preferential binding characteristics for the ligands in question.\n\n\nMethods\n\nProtein sequences were extracted from UniProt17. Many protein structure modeling methods have been developed and are available with most performing well given crystallographic template(s) sharing sufficient sequence homology with target sequences of interest18. The Eidogen StructFast19,20 technology is well suited for this type of modeling. StructFast can operate in an automated mode where the best PDB template is automatically selected, or in a directed mode where modeling is guided based on a suggested PDB template21,22.\n\nOnce models for A5UMI1 and Q02394 were developed with StructFast, ligand binding sites were identified by inference from the respective PDB templates used in modeling and from the Eidogen SiteSeeker algorithm23. SiteSeeker looks for concave, surface features sufficiently exposed to enable ligand binding while also considering evolutionary conservation of sequence. In addition to sites identified by SiteSeeker, other sites were manually inferred within PyMOL v1.8 after aligning models and templates containing their respective co-complexed ligands. Residues on model structures with a 7Å cutoff of co-complexed ligands within the templates were exported and also processed as sites.\n\nLigands were carefully prepared considering different protonation states, isomers, and tautomers. We standardized charges, added missing hydrogens, enumerated ionization states, ionized functional groups, generated tautomers and isomers, and generated starting-point 3D coordinates for each ligand using BIOVIA’s (Accelrys’) Pipeline Pilot technology v8.524. Ligands were finally prepared into mol2 format25. Each representation was then docked into each identified site and scored using AutoDock Vina v1.1.226, an open docking technology that utilizes grid-based energy evaluation and efficient search of ligand torsional freedom.\n\nThe AutoDock Vina system requires that receptor site files be formatted in the PDBQT [Protein Data Bank, Partial Charge (Q), & Atom Type (T)] molecular structure file format. The MGLTools v1.5.427 were used for this file format conversion. Additionally, AutoDock Vina requires a defined grid box surrounding the receptor site residues. Here, we identified the center of mass of each receptor site using all atoms in the receptor site PDB file. We then calculated within Pipeline Pilot the maximum distance between any atom in the receptor site and the centroid in each x,y,z-direction. The lengths of each grid box were configured with these maximums. To insure reproducibility and comparability of docking simulations, we initiated each AutoDock Vina run with the same random seed value of 1162467901.\n\n\nResults and discussion\n\n\n\nWe identified three different PDB templates that had sufficient sequence homology to model the Q02394 sequence, by identifying other PDB co-complexes containing ligands with high 2D similarity to the H4M ligand. The top three PDBs showing significant sequence homology to Q02394 included: 3F47 (57%), 3H65 (57%), and 4JJF (52%). Each template was used to model Q02394. In each case, ligand-binding sites were readily inferred from the ligand binding sites found in the respective template structures.\n\nThe modeling of sequence A5UMI1 was straightforward given its high 52% sequence homology to 3IQZ. Each 3IQZ chain (A-F) was considered, given the possibility that one template-chain might offer additional or different insight into possible ligand binding locations. The Eidogen SiteSeeker algorithm identified only one site when template chains A, C, D were used, while two sites were identified in models leveraging template chains B, E, F. Unfortunately, the H4M site from the 3IQZ template was not easily inferred into any of the A5UMI1/3IQZ-based models, because 3IQZ has multiple chains involved in H4M binding.\n\nModeling sequences from PDB templates is done with individual chains. Quaternary modeling using models of individual chains can be very challenging. We manually modeled the H4M site as described in Figure 1. Since our aim was to dock all ligands across all possible ligand binding sites, we included the sites identified by inference (i.e. where ligands were present in templates), by the SiteSeeker algorithm run across single chain models, and by manually modeled sites as described by Figure 1. A total of 10 ligand-binding sites (Table 1) were identified across all the Q02394 and A5UMI1 models.\n\nModeled quaternary structure of A5UMI1/3IQZB (cyan) and A5UMI1/3IQZF (pink) after respective alignments onto chain-B and chain-F of 3IQZ within PyMOL28. 3IQZ’s chain-F is highlighted in silver. Dual chain model site residues (blue surface) were inferred from residues in chain-B and chain-F models that are within 7 Å of the 3IQZ ligand (H4M - white). 3IQZ’s chain-B and chain-F form a quaternary structure with two different H4M binding sites (bottom).\n\nFour sites from the A5UMI1 modeling and six sites from Q02394 modeling were used in the docking simulations.\n\nThe key ligands for this effort included lovastatin (lactone and hydroxyacid forms), F420, and simvastatin (lactone and hydroxyacid forms) and processed ligands that were found in the PDB templates used to model each sequence: 803, F42, H4M, I2C, FE9, SIM, 116, HMG, and 882. The latter four (SIM, 116, HMG, 882) were ligands found in the positive control templates for completeness.\n\nThe PDB often contains problematic ligand structures, so we processed both PDB ligands and ligands extracted from PubChem29 for lovastatin (lactone and hydroxyacid forms), F420, and simvastatin (lactone and hydroxyacid forms). It should be noted, the PDB considers 803 as lovastatin (lactone form), F42 as coenzyme-F420, and SIM as simvastatin (hydroxyacid form), though their actual structural forms may vary depending on the PDB entry. This is why we also use PubChem structural representations for lovastatin, F420, and simvastatin.\n\nIt is well established that the β-hydroxyacid form and not the closed-ring lactone form of lovastatin is the active HMGR-binding form of the molecule30. Simvastatin and lovastatin are commercially available in the lactone form; they behave as prodrugs which inhibit HMGR only after the opening of the lactone ring into the hydroxyacid form31,32. The degree of hydrophobicity of imidazole derivatives correlates with improved activity against human methanogenic archaea33.\n\nEach ligand was computationally processed in the same way prior to docking. BIOVIA’s (Accelrys’) Pipeline Pilot was used for this ligand preparation. First, stereochemistry and charges were standardized, then ionized at pH 7.4, then tautomers (if present) were enumerated, and finally initial 3D models were determined. AutoDock Vina explores ligand 3D conformation, so the initial 3D models were simple starting points. Additionally, ligands were processed without the above standardization, ionization, and tautomer exploration. Each ligand representation was considered in the docking runs. Ligands processed with the standardization sequenced contained the prefix “STD_,” and ligands without standardization contained the prefix “RAW_.” Together, these expanded ligand representations can help gauge the docking algorithm’s sensitivity to the ligand’s structural representation.\n\nA total of 88 ligand variations were systematically docked into the 10 identified binding sites across all the A5UMI1 and Q02394 models for a total of 880 docking simulations. Even though AutoDock Vina achieves two orders of magnitude speed-up and significantly improves the accuracy of the binding mode predictions compared to AutoDock 4, 880 docking simulations could have taken several weeks to complete. To accelerate the effort, we requisitioned a compute cluster in the Amazon EC234 cloud environment for approximately three days at a cost under $60.\n\nThe docking process scores ligand conformations based on ligand conformation and ligand-to-receptor interactions within a grid box. After the 880 docking simulations were complete, we rescored all docked ligand variations against their respective full model structures. This enabled a more realistic rank ordering given possible overlap with a docked ligand and other portions of a model not represented in the rectangular box. This also served as an internal control, since rescoring was completed independently of the docking simulations.\n\nSince it is unknown which (if any site) might actually engage the ligands of interest, we calculated the average, minimum, and maximum affinity of each ligand/variation for each of the 10 sites. The top-two sites (highlighted in bold in Table 2) were used to then rank order each ligand. Table 3 shows the rank ordered ligands using the AutoDock Vina overall score, which considers steric interactions (Gauss 1, Gauss 2, and steric), dispersion/repulsion, hydrophobic interaction between hydrophobic atoms, and, where applicable, hydrogen bonding.\n\nAffinities were computed from AutoDock Vina26. The top two scoring sites from A5UMI1 and Q02394 are in bold. These sites were used to rank the ligands in Table 3.\n\nStatin ligands highlighted in green are lactone form, or red if hydroxyacid form. F420 ligands are in blue. Tautomeric representations are included in each average. Standardized ligands are prefixed with “STD_,” those without standardization are prefixed with “RAW_” (see text). Ligand names have suffixes containing either the PDB entry they were originally extracted from, or their respective PubChem29 CIDs.\n\nGiven the rank ordering in Table 3, several observations became evident:\n\n1) Consistent with Sharma et al.14, the lactone form statins docked into each site with favorable site interactions (i.e. lower docking scores) as compared to F420 for the same sequence/site grouping.\n\n2) The statin lactone forms generally had more favorable docking scores, even relative to the native template PDB ligands.\n\n3) The statin hydroxyacid forms had less favorable docking scores and typically scored in the middle with some of the F420 forms.\n\n4) The F420 scores were generally the lowest for each sequence/site models of A5UM1 and Q02394.\n\nTable 4 (a,b) details the AutoDock Vina scoring metrics of lovastatin-lactone v. lovastatin-hydroxyacid across the top two modeled sites. The lovastatin lactone form had better AutoDock scores across each site as compared to the hydroxyacid form. Similarly, the calculated affinity (kcal/mol) of the lactone form was better within both modeled A5UMI1 sites. Figure 2 depicts the best scoring lovastatin-lactone and –hydroxyacid poses in the A5UMI1 modeled site (top) and the Q02394 modeled site (bottom). The A5UMI1 modeled site is more spherically form fitting while the Q02394 modeled site is more elongated. The A5UMI1 site also contains a greater concentration of hydrophilic resides (depicted in cyan in Figure 2). In each modeled site, the best scoring lactone and hydroxyacid form were docked roughly in the same position with similar interactions, however the lactone form contained more favorable intermolecular feature.\n\nAutoDock4.1Score is a weighted sum of steric interactions (Gauss 1, Gauss 2, and steric), repulsion, hydrophobic interaction between hydrophobic atoms, and, where applicable, hydrogen bonding26.\n\nBest scoring lovastatin-lactone and -hydroxyacid poses in A5UMI1 3IQZB_SiteSeeker2 (top) and Q02394 4JJF_SiteSeeker (bottom). Lovastatin-lactone form is shown with green sticks and hydroxyacid form with red sticks. Residues within 5 angstroms of ligands are labeled. Hydrophilic site residues are shown in cyan and hydrophobic residues in gray.\n\nFigure 3 depicts lovastatin-lactone (top) v. F420 (bottom) docked into the top A5UMI1 modeled site (see Dataset 2 helps to visualize and perceive additional detail depicted). Lovastatin-lactone had better AutoDock scores and more favorable calculated affinities – despite having fewer hydrogen bond interactions. Both ligands appear to be interacting with ARG-255, ARG-150, and GLN-153, though F420 seems to also interact with ARG-244. F420’s fit is also considerably more constrained, which explains why its AutoDock Vina score is 4.4× worse than lovastatin-lactone’s score.\n\nLovastatin-lactone 1: (top) [Calculated affinity: -7.2 (kcal/mol); AutoDock4.1Score: 14.3]; 2: (bottom) F420 [Calculated affinity: -6.99 (kcal/mol); AutoDock4.1Score: 63.3] docked into A5UMI1_3IQZB_SiteSeeker2. Hydrogen bond interactions are denoted with yellow dotted lines.\n\n\nConclusions\n\nGiven the large number of ligand-to-site docking scenarios, we were able to observe several key trends that together suggest that statin binding is likely for the two key targets in question A5UMI1 and Q02394. In most cases, the lactone form appears to have preferential binding over the hydroxyacid form and F420. And in many cases, lovastatin/lactone and simvastatin/lactone appear to have preferential binding to even the native ligands found in the PDB templates used to model Q02394 and A5UMI1.\n\nThe docking simulations are consistent with those from a recent phase II clinical trial (NCT0249562335) with a proprietary, modified-release lovastatin-lactone (SYN-010) in patients with constipation-predominant, irritable bowel syndrome, which showed a reduction in symptoms and breath methane levels compared to placebo. Given that the lactone form seems to preferentially bind, the next stage of the project is to identify molecules with similar features to lovastatin-lactone that also show similar or better receptor-site interaction potential.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for ‘Lovastatin lactone may improve irritable bowel syndrome with constipation (IBS-C) by inhibiting enzymes in the archaeal methanogenesis pathway’, 10.5256/f1000research.8406.d11791736\n\nFigshare: Lovastatin-lactone v. F420 in the A5UMI1 site. doi: 10.6084/m9.figshare.3126538.v137",
"appendix": "Author contributions\n\n\n\nSM: Finalized analysis workflow, suggested additional hypotheses, performed the experiments, and wrote first and subsequent drafts of the manuscript.\n\nJS: Provided many helpful suggestions during the manuscript preparation.\n\nJK: Made many helpful suggestions during the initial planning and during manuscript review.\n\nMP: Provided insights into methane microbiology and reviewed all drafts.\n\nVW: Co-developed the research question and design of experiments and reviewed all drafts.\n\nKG: Originated the research idea, the major hypotheses and a draft analysis plan and reviewed all manuscript drafts.\n\nAll authors 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\nFacts About IBS. Reference Source\n\nSperber AD, Dumitrascu D, Fukudo S, et al.: The global prevalence of IBS in adults remains elusive due to the heterogeneity of studies: a Rome Foundation working team literature review. Gut. 2016; pii: gutjnl-2015-311240. PubMed Abstract | Publisher Full Text\n\nPimentel M, Gunsalus RP, Rao SSC, et al.: Methanogens in Human Health and Disease. Am J Gastroenterol Suppl. 2012; 1(1): 28–33. Publisher Full Text\n\nZhang H, Banaszak JE, Parameswaran P, et al.: Focused-Pulsed sludge pre-treatment increases the bacterial diversity and relative abundance of acetoclastic methanogens in a full-scale anaerobic digester. Water Res. 2009; 43(18): 4517–26. PubMed Abstract | Publisher Full Text\n\nMiller TL, Wolin MJ: Methanogens in human and animal intestinal Tracts. System Appl Microbiol. 1986; 7(2–3): 223–9. Publisher Full Text\n\nJain S, Caforio A, Driessen AJ: Biosynthesis of archaeal membrane ether lipids. Front Microbiol. 2014; 5: 641. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiller TL, Wolin MJ: Inhibition of growth of methane-producing bacteria of the ruminant forestomach by hydroxymethylglutaryl-SCoA reductase inhibitors. J Dairy Sci. 2001; 84(6): 1445–8. PubMed Abstract | Publisher Full Text\n\nMarsh E, Morales W, Chua KS, et al.: Lovastatin Lactone Inhibits Methane Production in Human Stool Homogenates. Reference Source\n\nJahromi MF, Liang JB, Ho YW, et al.: Lovastatin production by Aspergillus terreus using agro-biomass as substrate in solid state fermentation. J Biomed Biotechnol. 2012; 2012: 196264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu J, Zhang J, Shi Y, et al.: Chinese red yeast rice (Monascus purpureus) for primary hyperlipidemia: a meta-analysis of randomized controlled trials. Chin Med. 2006; 1: 4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhao ZJ, Pan YZ, Liu QJ, et al.: Exposure assessment of lovastatin in Pu-erh tea. Int J Food Microbiol. 2013; 164(1): 26–31. PubMed Abstract | Publisher Full Text\n\nPérez-Gil J, Rodríguez-Concepción M: Metabolic plasticity for isoprenoid biosynthesis in bacteria. Biochem J. 2013; 452(1): 19–25. PubMed Abstract | Publisher Full Text\n\nGottlieb K, Wacher V, Sliman J, et al.: Review article: inhibition of methanogenic archaea by statins as a targeted management strategy for constipation and related disorders. Aliment Pharmacol Ther. 2016; 43(2): 197–212. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSharma A, Chaudhary PP, Sirohi SK, et al.: Structure modeling and inhibitor prediction ofNADP oxidoreductase enzyme from Methanobrevibacter smithii. Bioinformation. 2011; 6(1): 15–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFerry JG: Enzymology of one-carbon metabolism in methanogenic pathways. FEMS Microbiol Rev. 1999; 23(1): 13–38. PubMed Abstract | Publisher Full Text\n\nProtein Data Bank archive (PDB). Reference Source\n\nUniProt Consortium: UniProt: a hub for protein information. Nucleic Acids Res. 2015; 43(Database issue): D204–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nList of protein structure prediction software. Reference Source\n\nDebe DA, Danzer JF, Goddard WA, et al.: STRUCTFAST: protein sequence remote homology detection and alignment using novel dynamic programming and profile-profile scoring. Proteins. 2006; 64(4): 960–7. PubMed Abstract | Publisher Full Text\n\nPoleksic A, Danzer JF, Hambly K, et al.: Convergent Island Statistics: a fast method for determining local alignment score significance. Bioinformatics. 2005; 21(12): 2827–31. PubMed Abstract | Publisher Full Text\n\nPalmer B, Danzer JF, Hambly K, et al.: StructSorter: a method for continuously updating a comprehensive protein structure alignment database. J Chem Inf Model. 2006; 46(4): 1871–6. PubMed Abstract | Publisher Full Text\n\nHambly K, Danzer J, Muskal S, et al.: Interrogating the druggable genome with structural informatics. Mol Divers. 2006; 10(3): 273–81. PubMed Abstract | Publisher Full Text\n\nEidogen-Sertanty SiteSeeker. Eidogen-Sertanty, Inc. Reference Source\n\nBIOVIA Pipeline Pilot. Reference Source\n\nTripos Mol2 File Format. Reference Source\n\nTrott O, Olson AJ: AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010; 31(2): 455–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDallakyan S: MGLTools. Reference Source\n\nSchrodinger LLC: The PyMOL Molecular Graphics System. Version 1.8. 2015. Reference Source\n\nKim S, Thiessen PA, Bolton EE, et al.: PubChem Substance and Compound databases. Nucleic Acids Res. 2016; 44(D1): D1202–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDuggan DE, Chen IW, Bayne WF, et al.: The physiological disposition of lovastatin. Drug Metab Dispos. 1989; 17(2): 166–73. PubMed Abstract\n\nSirtori CR: The pharmacology of statins. Pharmacol Res. 2014; 88: 3–11. PubMed Abstract | Publisher Full Text\n\nWood WG, Mΰller WE, Eckert GP: Statins and neuroprotection: basic pharmacology needed. Mol Neurobiol. 2014; 50(1): 214–20. PubMed Abstract | Publisher Full Text\n\nKhelaifia S, Brunel JM, Raoult D, et al.: Hydrophobicity of imidazole derivatives correlates with improved activity against human methanogenic archaea. Int J Antimicrob Agents. 2013; 41(6): 544–7. PubMed Abstract | Publisher Full Text\n\nAmazon Elastic Compute Cloud (Amazon EC2). Reference Source\n\nGottlieb K: NCT02495623 - A Study of the Effect of SYN-010 on Subjects With IBS-C. In: Inc. SB, editor. 2015. Reference Source\n\nMuskal S, Sliman J, Kokai-Kun J, et al.: Dataset 1 in: Lovastatin lactone may improve irritable bowel syndrome with constipation (IBS-C) by inhibiting enzymes in the archaeal methanogenesis pathway. F1000Research. 2016. Data Source\n\nMuskal S, Sliman J, Kokai-Kun J, et al.: Lovastatin-lactone v. F420 in the A5UMI1 site. Figshare. 2016. Data Source"
}
|
[
{
"id": "13313",
"date": "11 Apr 2016",
"name": "Rolf Thauer",
"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 describes modeling studies suggesting that Lovastin lactone, a statin, inhibits growth of methane-forming archaeon Methanobrevibacter smithii by inhibiting an F420 dependent-enzyme involved in CO2 reduction to methane, namely F420-dependent methylene-tetrahydromethanopterin dehydrogenase (Mtd). Modeling studies had previously indicated that another F420-dependent enzyme, F420H2:NADP oxidoreductase in methanogens could be a site of inhibition (reference 14). The results are interesting, however, before indexing the following information has to be added: The authors must provide experimental evidence that their theoretical prediction is correct. Show that Lovastin inhibits methane formation from H2 and CO2 in non-growing cell suspensions of M. smithii and/or that Lovastin inhibits Mtd activity in cell extracts of M. smithii of better with the purified enzyme. The authors might want to team up with a lab experienced in the proposed experiments. Without these experimental data the manuscript would contain nothing really new relative to the results published in reference 14. The authors must clearly indicate that crystal structures of Mtd with and without substrates bound have been published and give reference to these publications. To only refer to PDBs without the enzyme name is not fair. To indicate in the Abstract, Methods, that there is “no tertiary protein structural information” is more than misleading and can be misunderstood.",
"responses": [
{
"c_id": "1927",
"date": "28 Apr 2016",
"name": "Steven Muskal",
"role": "Author Response",
"response": "Thank you for your review and comments.Based on your comments we fixed the misleading text in the Abstract that you noted, and also corrected a typographical swapping of the sequence IDs in the introduction.We originally did not feel it necessary to mention all the possible templates considered in the modeling effort, but appreciate the value of doing so. Per your suggestion, we inserted additional text in the modeling section which refers to other relevant PDB templates - e.g. 1QV9 and 1U6I,J,K. 1QV9 - Coenzyme F420-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) from Methanopyrus kandleri: A methanogenic enzyme with an unusual quaternary structure, and 1U6I,J,K - The structures of native coenzyme F420-dependent methylenetetrahydromethanopterin dehydrogenase at various resolutions, and TLS refinement of the structure of Se-methionine labelled Coenzyme f420-dependent methylenetetrahydromethanopterin dehydrogenase (MTD) from Methanopyrus kandleri. With respect to the experimental data comment, while this paper is a computational chemistry paper, we did note and reference the recently completed clinical trial - A Study of the Effect of SYN-010 on Subjects With IBS-C (https://clinicaltrials.gov/ct2/show/NCT02495623) as well as experimental work by Marsh et al. - Lovastatin Lactone Inhibits Methane Production in Human Stool Homogenates (http://content.stockpr.com/syntheticbiologics/db/220/608/file/Lovastatin+in+Human+Stool-FINAL+Poster+%28ACG+2015%29.pdf). In the latter, albeit a messy culture, M. smithii was the predominant methanogen in the stool, though the less common Methanosphaera stadtmanae may have been present as well."
}
]
}
] | 1
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https://f1000research.com/articles/5-606
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https://f1000research.com/articles/5-1462/v1
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22 Jun 16
|
{
"type": "Case Report",
"title": "Case Report: Subacute onset of the motor-sensory axonal neuropathy variant of Gullain-Barré syndrome after epidural anesthesia",
"authors": [
"Marie-Louise Kulas Søborg",
"Jacob Rosenberg",
"Jakob Burcharth",
"Jacob Rosenberg",
"Jakob Burcharth"
],
"abstract": "Guillain-Barré syndrome (GBS) is an acute ascending peripheral neuropathy, caused by autoimmune damage of the peripheral nerves. GBS can be divided into three subtypes: acute inflammatory demyelinating neuropathy, acute motor axonal neuropathy, and the more rare type, acute motor and sensory axonal neuropathy (AMSAN). Reports of AMSAN with onset after epidural anesthesia and spinal surgery are extremely rare, and the linkage between development of GBS and neuroaxial anesthesia remains conclusively unconfirmed. We present a case in which the patient developed subacute motor and predominantly sensory neuropathy following epidural blockade. The case emphasizes the need of including AMSAN in differential diagnostic considerations to changes in motor and sensory function following epidural anesthesia, allowing accelerated rehabilitation and relevant alleviating therapy.",
"keywords": [
"Guillain-Barré syndrome",
"AMSAN",
"neuroanesthesia",
"epidural blockade"
],
"content": "Background\n\nGuillain-Barré syndrome (GBS) is an acute peripheral neuropathy caused by an autoimmune response against the myelin sheets of peripheral nerves and can be subdivided into motor and mixed motor and sensory types1,2. Acute onset of GBS after epidural anesthesia has previously been reported, raising concern of a correlation between GBS and use of neuroaxial anesthesia3,4.\n\nIn this case report a patient is presented with subacute onset of motor and predominantly sensory polyneuropathy following epidural anesthesia.\n\n\nCase\n\nA 65-year-old woman without previous neurological disease was admitted to the surgical department due to pain in the sacral bone and one and a half week lasting water-thin stool per rectum and brownish stool per vaginam. The patient had various comorbidities, including previous left-sided nephrectomy, hysterectomy, and cystectomy with an ileum bladder due to stage T4 vesical cancer. The patient had no history of recurrence.\n\nComputed tomography (CT) and magnetic resonance imaging (MRI) confirmed sigmoid diverticulosis with diverticular stenosis and fistulae between colon-rectum and upper vagina. A Hartmann´s procedure was performed with combined general and epidural anesthesia and the epidural infusion was discontinued after 2 days. One day after discontinuation, the patient developed dysesthesia and numbness in both hands and lower extremities up to knee level, accompanied by substantial decrease of fine motor skills. Symptoms were initially interpreted as unintended neural compression during surgery.\n\nNeurological examination 10 days after onset of symptoms showed unaffected cerebral function with preserved function of all cranial nerves. Both hands presented with symmetrical, bilateral atrophy. Motor testing showed grade 4+ muscle strength on a 1–5 muscle strength scale in all extremities, with all deep tendon reflexes preserved, except for the Achilles bilaterally. Sensory testing confirmed dysesthesia ascending up to wrist- and knee level bilaterally with reduced sense of touch, vibration, pain, and thermal changes. Proprioception remained intact but the patient was observed with ataxic walk. The condition was determined as a motor and predominantly sensory polyneuropathy with subacute onset and unknown etiology. A lumbar puncture found inconspicuous cerebrospinal fluid with normal protein level and absent pleocytosis. Electromyography confirmed a sensorimotor polyneuropathy in upper and lower extremities of predominantly axonal type. Columnar CT and MRI revealed anatomical compression with bilateral S1 root compression and multiple segments presenting anterolistesis. However, the specialized unit for columnar diseases found neither any plausible cause for the current symptoms, nor indication for decompressive surgery. Positron emission tomography–computed tomography disproved plausible neoplastic cause for the neuropathy. Paraclinically, the patient had a normal biochemical profile except for an increased erythrocyte sedimentation rate and an increased level of light-chain immunoglobulins, subsequently discarded as possible light-chain amyloidosis by negative Congo-red coloration.\n\nThe patient was only treated with gabapentin, parenteral nutrition and substitution of vitamins B, D and magnesium during the next ten months after onset of neurological symptoms. Parental nutrition was given for one week shortly after onset of neurological symptoms. Vitamin B was substituted through intramuscular injection of 1 milligram (mg) every third month, vitamin D by two tablets of 25 mg daily and magnesium by one tablet of 360 mg daily. Pain relief was provided by an initial dosage of 300 mg gabapentin daily, and increased intermittently during the following one and a half year to 900 mg three times daily.\n\nThe patient was followed every third month at the Neurology clinic for the next one and a half year. Her condition remained unchanged with stationary paresthesia in upper and lower extremities, considerably impaired activity-level, and complicated by neuropathic pain.\n\n\nDiscussion\n\nThis case report followed the CARE Guidelines for reporting of case reports5 and details of surgery, hospitalization and physical examinations were retrieved from the official national registration system used at all Danish hospitals, with consent from the patient.\n\nGBS can be divided into three subtypes: acute inflammatory demyelinating neuropathy, acute motor axonal neuropathy, and the more rarely seen acute motor and sensory axonal neuropathy (AMSAN)6. The majority of cases with GBS are caused by prior infection of the upper respiratory tract or gastroenteritis and a subsequent abnormal immune response to the infectious agent, typically with onset of symptoms 2–4 weeks after the initial infection2,6.\n\nOur case presented a subacute motor and predominantly sensory affection, most consistent with the AMSAN subtype. This was supported by the consistent findings at the neurological examination and electromyography. Differential diagnoses were inflammatory neuropathy or neuropathy due to mineral- or tracer deficiency caused by diarrhea or previous small intestine resection. The patient was substituted with vitamins B, D and magnesium without remission of the current complaints. Cerebrospinal fluid and biochemical parameters showed no signs of cerebral infection and the patient did not present with fever. Although diarrhea was present, this debuted prior to onset of neurological symptoms and whether the diarrhea was on an infectious background was undetermined, but it could most likely be explained by her complicated diverticular disease. Furthermore, diagnostic imaging revealed no relevant anatomical pathology causing the neurological symptoms. Thus, the patient presentation and clinical course were highly suggestive of AMSAN.\n\nPrior cases of acute onset of AMSAN after spinal surgery or epidural anesthesia have been reported4,6,7. The suggested underlying mechanism is probably an intraoperative release of antigens, leading to autoimmunization and a subsequent immune response with attack of neural elements4,6. Other theories have suggested how surgery may cause either an immune imbalance with increased number of T-cells, or a transient immunosuppression promoting subclinical infections4. The AMSAN variant may result in fulminant weakness and sensory loss within 7 days, and recovery is poor8,9 in contrast to the typical GBS presentation with motor impairment that typically will recur fully within weeks to months2. No curative interventions are currently known, but this case emphasizes the importance of aggressively investigating any new onset of motor and sensory loss postoperatively since it may be the AMSAN variant of GBS. This could possibly aid to accelerate correct diagnosis and forward rehabilitation and relevant alleviating therapy with considerable effect on the patients’ quality of life.\n\nThis case report presents a case of subacute motor and predominantly sensory loss after epidural anesthesia, highly suggestive of AMSAN. Although rarely reported, AMSAN should be included in the differential diagnostic considerations to changes in motor or sensory function after epidural or spinal anesthesia.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for publication of this case report and any accompanying images and/or other details that could potentially reveal the patient’s identity.",
"appendix": "Author contributions\n\n\n\nM.S.: study design, drafting, and final approval; J.R: study design, revision, and final approval; J.B.: study design, revision, and final approval.\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\nYuki N, Hartung HP: Guillain-Barré syndrome. N Engl J Med. 2012; 366(24): 2294–304. PubMed Abstract | Publisher Full Text\n\nvan den Berg B, Walgaard C, Drenthen J, et al.: Guillain-Barré syndrome: pathogenesis, diagnosis, treatment and prognosis. Nat Rev Neurol. 2014; 10(8): 469–82. PubMed Abstract | Publisher Full Text\n\nMangar D, Sprenker C, Karlnoski R, et al.: Rapid onset of guillain-barré syndrome after an obstetric epidural block. A A Case Rep. 2013; 1(1): 19–22. PubMed Abstract | Publisher Full Text\n\nBoghani Z, Livingston AD, Simpson EP, et al.: Acute Onset of Guillain-Barré Syndrome After Elective Spinal Surgery. World Neurosurg. 2015; 84(2): 376–9. PubMed Abstract | Publisher Full Text\n\nGagnier JJ, Kienle G, Altman DG, et al.: The CARE guidelines: consensus-based clinical case report guideline development. J Clin Epidemiol. 2014; 67(1): 46–51. PubMed Abstract | Publisher Full Text\n\nCheng J, Kahn DE, Wang MY: The acute motor-sensory axonal neuropathy variant of Guillain-Barré syndrome after thoracic spine surgery. J Neurosurg Spine. 2011; 15(6): 605–9. PubMed Abstract | Publisher Full Text\n\nSteiner I, Argov Z, Cahan C, et al.: Guillain-Barré syndrome after epidural anesthesia: direct nerve root damage may trigger disease. Neurology. 1985; 35(10): 1473–5. PubMed Abstract | Publisher Full Text\n\nKoc M, Ozalp N, Zulfikaroglu B: Major surgery with Guillain-Barré syndrome: a case report. J Int Med Res. 2002; 30(6): 601–4. PubMed Abstract | Publisher Full Text\n\nShah DN: The spectrum of Guillain-Barré syndrome. Dis Mon. 2010; 56(5): 262–5. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "15393",
"date": "17 Aug 2016",
"name": "Jean-Michel Vallat",
"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, but I am not sure that at the present time, we have enough data; if the authors can let us know the followings, it will be all right:\nWhy Campylobacter jejuni or any other infection was not looked for, as the diarrhea was present prior to the onset of the neurological symptoms? Were anti-glycolipid antibodies were tested? In a significant number of acute dysimmune neuropathies, they are considered as useful biomarkers if they are present. If AMSAN was discussed, why did the patient did not receive at the beginning, any treatment such as immunoglobulins or plasma exchanges?\nI am surprised that after 10 days of evolution, when the motor testing was only 4+, a bilateral atrophy of hands was observed.\nActually, the first AMSAN cases have been reported by Feasby et al. (1986).",
"responses": []
},
{
"id": "15395",
"date": "18 Aug 2016",
"name": "Gerard Said",
"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\nInteresting case of an \"axonal GBS\" syndrome occurring shortly after epidural anesthesia, in a patient with a number of health problems. I assume that the patient could also have developed a nutritional axonal polyneuropathy due to her poor health condition.\n\nThe authors claim that light chain amyloidosis was excluded after negative Congo red staining. Which tissue was studied ?\nAmyloid neuropathy unlikely because of the rate of onset, and stability of deficit afterwards.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1462
|
https://f1000research.com/articles/4-798/v1
|
14 Sep 15
|
{
"type": "Research Article",
"title": "Twittering About Research: A Case Study of the World’s First Twitter Poster Competition",
"authors": [
"Edward P. Randviir",
"Samuel M. Illingworth",
"Matthew J. Baker",
"Matthew Cude",
"Craig E. Banks",
"Edward P. Randviir",
"Samuel M. Illingworth",
"Matthew J. Baker",
"Matthew Cude"
],
"abstract": "The Royal Society of Chemistry held, to our knowledge, the world’s first Twitter conference at 9am on February 5th, 2015. This paper reports the details of the event and discusses the outcomes, such as the potential for the use of social media to enhance scientific communication at conferences. In particular, the present work argues that social media outlets such as Twitter broaden audiences, speed up communication, and force clearer and more concise descriptions of a researcher’s work. The benefits of poster presentations are also discussed in terms of potential knowledge exchange and networking. This paper serves as a proof-of-concept approach for improving both the public opinion of the poster, and the enhancement of the poster through an innovative online format that some may feel more comfortable with, compared to face-to-face communication.",
"keywords": [
"Twitter",
"Research",
"Poster",
"Competition",
"Engagement",
"Communication",
"Chemistry",
"Conference"
],
"content": "Introduction\n\nAn academic conference should be a symposium where academics can report, share, discuss their work, and exchange ideas through a variety of different communication methods. A typical academic conference may consist of several oral presentations, including those from keynote or plenary speakers, in addition to a number of workshops, which offer a more interactive method of delivery. There is also the research poster, a somewhat maligned and misunderstood entity that in some instances feels like an afterthought. However, it could be argued that if knowledge exchange is the fundamental purpose of a conference, which it is, then posters and workshops are far more valuable than oral presentations (Rowe & Ilic, 2009); in which case, are posters being unfairly discredited?\n\nThe “all eyes on one” style of oral presentations is extremely limiting in terms of opportunities for the speaker to interact personally with members of the audience. Even in the post-talk questions, time constraints mean that not all questions can be asked, whilst some participants may be unable to comfortably relay their points to the author in that particular environment. Aside from this, the less intimidating nature of poster sessions may be preferable to a larger percentage of researchers, which might explain why the poster presentation saw large increases in the 1990s (Moule et al., 1998).\n\nA poster session is an extended period of academic knowledge exchange. Exhibitors normally stand by their poster and explain their research and findings to passing delegates, inspiring some form of discussion as a dialogue or perhaps in a group. Therefore, it makes sense poster sessions should provide more frequent opportunities for academics to exchange knowledge and create networks. The format of a poster session should theoretically allow for open, informal, and comfortable academic discussion regarding the work presented. Many researchers will have experienced instances of such academic exchanges taking place; yet it is not a form of communication that has been formally investigated in any great detail, but for contributions from Dubois Betty (1985) and Shalom (1993), who independently suggest that the poster presentation was a genre struggling for definition some 20 years ago; the feeling is unfortunately similar within scientific disciplines today. However, it has also been shown that students presenting posters on sensitive topics found that the format of the poster session put them more at ease (Rush et al., 1995). Such an observation would suggest that the poster acts as a message board and focal point for presenters, with sensitive topics such as sexuality made easier to discuss by using posters as a facilitator. This facilitatory role can be extended to other less taboo-orientated subjects and, in principle, the poster could help to facilitate learning amongst researchers, especially those in the early stages of their careers who may be less confident when presenting their research, compared to other, more experienced colleagues.\n\nDespite many efforts by academics to report good poster guidelines (see e.g. Erren & Bourne, 2007; Hess et al., 2009; Moore et al., 2001; Shelledy, 2004; Taggart & Arslanian, 2000), the ideal poster presentation is often absent from poster sessions. Many posters are either poorly designed, or simply pinned to boards and left to stagnate, leaving any observant or enthusiastic researchers with unanswered questions. Even if a poster manages to attract a delegate, the content must be written in a concise, clear, and jargon-free manner to inspire intrigue. Poor written communication can be as detrimental to the message as the oral communication blunders brought about by an ill-prepared delivery.\n\nIt therefore comes as no surprise that some organisations have attempted to reimagine the poster. One specific example of this comes from the European Geosciences Union (EGU), who use a concept called PICO (Presenting Interactive COntent) to diversify the knowledge exchange process. The general idea of PICO is for researchers to orally advertise their work in a two-minute flash presentation, in order to encourage the audience to later join them at interactive touchscreen slides, where they can engage with the author personally, in a format similar to the traditional poster session (European Geosciences Union, 2015). Such a form of engagement will no doubt enhance the learning and knowledge exchange experience for the researcher.\n\nWith any conference, there are always academics wishing to participate, but who are unable to because of travel and funding restrictions. For some researchers, these restrictions can be detrimental for the dissemination of their research, and can ultimately have a negative effect on their career progression. To combat this, some organisations, like the American Geophysical Union (AGU), have piloted a virtual poster showcase, encouraging researchers to participate at conferences virtually through a digital link. This obviates the requirement for travel, and therefore extra funding for travel purposes. Furthermore, posters are becoming an ever more acceptable route into publication, via academic journals such as in F1000Research , which publishes posters and slides alongside more traditional articles, as a means of reference-worthy academic literature.\n\nAnother potential alternative is the use of social media to encourage poster engagement, and this route will form the focus of this paper. The ubiquity of social media is responsible for many of the social behaviours and patterns that have emerged as a result of online communication, and given the power of social media, it could potentially be harnessed to help ensure posters are more greatly discussed, thereby helping to improve ideas and knowledge exchange between academics. This paper presents findings from the world’s first Twitter poster conference, organised by the Royal Society of Chemistry, and discusses the potential impact of social media upon the academic poster.\n\n\nMaterials and methods\n\nThe Analytical Science Twitter Poster Conference (ASTPC) was organised by the Royal Society of Chemistry (RSC) journals Analyst, Analytical Methods and Journal of Analytical Atomic Spectroscopy (JAAS). The ASTPC took place from 9am on 5th February 2015 to 9am on the 6th February 2015, giving researchers a period of 24 hours to tweet pictures of their poster using the hashtag #RSCAnalyticalPoster. The aim of the ASTPC was to create an opportunity for participants to showcase their research, meet new people, share ideas and learn about some of the latest developments in different areas of analytical science. The conference was open to anyone working in any area of analytical science whose research topic was within the scope of Analyst, Analytical Methods, or JAAS.\n\nParticipants were encouraged to tweet their work, and to be available to answer any questions that interested academics (or indeed members of the general public) might have about their research. There were also prizes for the best Twitter poster, as judged by the scientific committee, with remuneration in the form of an iPod and RSC book vouchers. Furthermore, unlike a regular conference that charges fees to participate, this event was entirely free, and had no registration process other than an email to the journal to verify identity. A scientific committee consisting of 12 academics associated with the RSC were heavily involved in asking questions, generating discussions, and judging posters. Further information regarding the event can be found on the journal’s official blog (http://blogs.rsc.org/an/2014/12/19/rscanalyticalposter/).\n\nThis study was carried out according to the British Educational Research Association’s (BERA) ethical guidelines for educational research, with all of the data in this study fully anonymised. All work was also carried out according to the terms of use as indicated by Twitter's policies.\n\nThe participants that took part in the ASTPC are now assessed in terms of the number of tweets, area of the world from which the tweet was sent, total number of followers, and potential viewing audience for the tweets. All data is sourced from an online data collection program, available at http://www.followthehashtag.com. The data sample was taken over a period of 63 days, from 9am on 19th December 2014 to 9am on 20th February 2015. The data was collected from such an early date because this is when the initial announcement of the hashtag was made and promotion of the event began, however the vast majority of tweets were sent during the 24-hour window of the competition itself. Data collection stopped shortly after the prize winners were announced. Only tweets with the hashtag #RSCAnalyticalPoster were considered for the analysis, and so any figures reported here are most likely an underestimate, precluding any tweets for which the hashtag was absent. All reported times are in Greenwich Meantime.\n\n\nResults and discussion\n\nFigure 1 depicts a world map with the locations of persons that contributed to the ASTPC. Over 80 posters were submitted from Argentina, Australia, Brazil, Canada, Ireland, Italy, Mauritius, the UK, and the USA, with the highest number of contributors coming from European countries. From the diversity shown in Figure 1, it can be inferred that social media can be used to improve the accessibility of the poster by making it freely accessible across the world in a matter of minutes. This further presents opportunities for researchers to exchange comments in the form of tweets, a format that is designed to be both clear and concise. Such communiqués encourage researchers to think more directly about their research, as they must communicate their point in 140 characters or less. This concise form of communication could help both students and academics to communicate more effectively, particularly students who sometimes struggle to differentiate between description and analysis (Chanock, 2000).\n\nYellow points indicate multiple contributions, whilst blue data points indicate singular or near-singular contributions. Reproduced from data reports obtained from the website http://www.followthehashtag.com.\n\nTable 1 presents the statistics that were published following the ASTPC. During the designated time period, over 1700 tweets were sent with the hashtag #RSCAnalyticalPoster, originating from 378 different contributors. Each participant contributed 4.59 tweets on average to the discussion, with the total number of followers for each person that tweeted amounting to over 380,000. On average, every poster potentially received in excess of 4500 views from several areas across the world (see Figure 1).\n\nTotal tweets - the total number of tweets which included #RSCAnalyticalPoster, this includes retweets.\n\nTotal audience - the number of people who may have seen #RSCAnalyticalPoster in their Twitter feed. Calculated using the sum of followers from each contributor.\n\nContributors - number of unique Twitter accounts that used #RSCAnalyticalPoster.\n\nTotal impressions - the sum of contributor followers multiplied by the number tweets in which a contributor used #RSCAnalyticalPoster.\n\nFigure 2 displays a tweet and reach timeline that illustrates the frequency of activity across the 63 days of data collection. It is evident that there are two major zones of activity, as one would largely expect: the first when the conference was officially held, and the second when the prize winners were announced. The biggest reach and number of tweets was observed during the main event, followed by the prize announcements. Figure 2 also demonstrates that the majority of the tweets took place during the ASTPC itself, relating to the scientific posters rather than to advertising and promotion of the conference.\n\nGiven the nature of a Twitter discussion, it is perhaps more useful to present data relating to the number of contributions that users made as a whole, rather than as an average. Figure 3 depicts the individual contributions by author, and it is apparent that over half of the tweeters made only one contribution to the competition (200 users). More encouragingly, over 20% of contributors tweeted five or more times, and almost 10% of the contributors tweeted more than ten times. Indeed, the latter statistic infers that at least some useful exchanges were being made, even if it is difficult to gauge from such data how successful the exchanges may have been. The overall reach of each individual is difficult to estimate from such a dataset. One contributor may have contributed ten tweets to the discussion that has 300 people contributing, for example, yet only have ten followers, giving an overall reach value of around 310 people. Conversely, one person may have 1000 followers yet only contribute one tweet, yet their reach would be around 1300 people for one tweet alone. Therefore, it is fair to assume that Twitter can have a larger impact if the user has more followers, regardless of the number of contributions.\n\nReproduced from data reports obtained from the website http://www.followthehashtag.com.\n\nAnother important piece of information relates to the gender distribution at the Twitter conference. According to the RSC membership department, 27.7% of their members are female and 72.3% male, representing an uneven distribution of members by gender. Figure 4 displays the contributions of the ASTPC by gender, with 25.6% of contributions made by females and 74.4% by males. The fact that there is no significant difference between the RSC’s overall membership and the contributors at this event shows that the social media format is not conducive to stimulation of more or less average contributions based upon the gender of the participant. The level of participation in terms of registrants was different to this, however, as 59.6% of the registrants were female. Therefore, whilst a large proportion of females were willing to engage with the competition (a significantly higher proportion than would be expected based on the RSC membership), the discussions appeared to be dominated by male contributors.\n\nThe ASTPC was organised as a free event to encourage the sharing and exchange of knowledge through the use of social media. This pilot scheme saw a potential Twitter audience of over a quarter of a million people, demonstrating that posters can quite easily be shared using Twitter, to potentially reach thousands of times more people than they could at even the largest of international scientific conferences. Every day millions of people across the world access Twitter, new Twitter connections are being made, and opinion and discussion is stimulated as a result, while the introduction of hashtags has only served to group discussions together and augment the potential reach of a niche discussion. Even without an organised hashtag or event, a poster can have a larger potential audience than it would at a conference, where the audience will, at the very most, be a few hundred people. The number of useful exchanges between participants is less easy to enumerate, as one cannot quantify the level of interaction between academics in a given poster session and compare it to the number of tweets. A face-to-face conversation about a poster that is in front of two researchers will no doubt be more fulfilling in terms of knowledge exchange, because the conversation is not limited to a few characters. In the Twitter conference, there was an average of 4.5 tweets per person, suggesting that the level of academic discussion was somewhat limited. However, this is not to say that knowledge was not exchanged, but simply that the discussion part of the ASTPC may have been shorter than that of a standard poster session at a conference. This does not account for exchanges that may have been made in private, via emails or direct messaging facilities on Twitter.\n\nAs a concept, the Twitter poster conference has some definite advantages over a more traditional poster format, with the data analysed in this study supporting the notion that it is an extremely useful way of broadening the reach and potential audience of a poster. Another advantage is the ease of knowledge exchange for those who lack the confidence or interpersonal skills required for efficient face-to-face communications. It is also apparent that Twitter can decrease the cost of the poster to the researcher because it does not need to be carried as supplementary luggage during air travel; it also avoids potentially exorbitant printing fees at conferences for those who have lost or previously been unable to print their poster. Furthermore, the carbon footprint of a Twitter-only conference is extremely low (unquantified), whereas an international conference will exhibit a substantial carbon footprint, mainly due to air travel. Research by MMU (unpublished report, Jonathan Davies and Professor Callum Thomas) has recently found that an international conference of 178 delegates resulted in the equivalent of 177 tonnes of CO2 being produced, the majority of which came from the 1.25 million kilometres of air travel required for delegates to travel to the conference.\n\nThe nature of Twitter means that more in-depth forms of communication are limited through online exchanges, which could be seen as a disadvantage of the format. However, after the initial exchanges the delegate has the opportunity to extend any interactions further. This can be achieved by the exchange of emails, phone numbers, and Skype IDs for example, or in private messaging facilities over Twitter, meaning that more in-depth chats about the research in question can still be facilitated. The lack of interpersonal communication is disadvantageous, but should not detract researchers from a Twitter conference. It could be used alongside the traditional poster session, or as a separate entity of its own if a researcher is unable, or prefers not, to travel. Given Twitter’s recent foray into video streaming, the concept could also be adapted to include oral communications, in which researchers could tweet short video vignettes of their work, or even use Periscope to live-stream the entire presentation. The Twitter format could also potentially be used as a hybrid with the PICO concept discussed in the introduction.\n\nIn specific relation to the RSC, the results presented here indicate that whilst Twitter could be used as a tool to address the gender inequality, more needs to be done to encourage female participants to participate in the active Twitter discussions.\n\n\nConclusions\n\nThe world’s first Twitter conference could be considered a success in terms of potential audience, ease of knowledge exchange, lack of travel requirement, and reduction in carbon footprint. The conference reached out to many researchers across the world, and created an opportunity for participants to share their work not only with academics, but also with other interested parties such as writers, industries, friends and family, and even policy makers. Over 80 posters were tweeted with the hashtag #RSCAnalyticalPoster, reaching an audience potentially as large as 375,000 people, and the format of a Twitter poster conference has the potential to allow for research to be shared more quickly and cheaply, and in a more environmentally friendly manner. Despite some potential issues relating to prolonged exchanges, there is no doubt that the hybridisation of the academic conference and social media is something that could and should be seen more regularly in the future. We expect the use of social media to significantly expand scientific conferences due to the advantages identified above, and also to be utilised alongside conferences where physical participation occurs. The benefits of social media can help researchers organise their poster viewings at large conferences, for example future ACS conferences, helping to potentially improve the poster session experience for all participating researchers. Such an improvement in engagement will enhance scientific communication and knowledge exchange, ultimately leading to more successful conferences.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for ‘Twittering About Research: A Case Study of the World’s First Twitter Poster Competition’., 10.5256/f1000research.6992.d101516 (Randviir et al., 2015).\n\nAll data is also available publically by searching for the hashtag “#RSCAnalyticalPoster” on FollowtheHashtag.",
"appendix": "Author contributions\n\n\n\nAll authors have contributed to this article equally.\n\n\nCompeting interests\n\n\n\nThe authors declare that there are no competing interests, either financial or otherwise.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to thank the Royal Society of Chemistry for access to member information and hosting the event.\n\n\nReferences\n\nChanock K: Comments on Essays: Do students understand what tutors write? Teaching in Higher Education. 2000; 5(1): 95–105. Publisher Full Text\n\nDubois BL: Popularization at the highest level: poster sessions at biomedical meetings. Int J Sociol Lang. 1985; 1985(56): 67–84. Publisher Full Text\n\nErren TC, Bourne PE: Ten simple rules for a good poster presentation. PLoS Comput Biol. 2007; 3(5): e102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEuropean Geosciences Union: PICO - Presenting Interactive COntent. [Online]. 2015. Reference Source\n\nHess GR, Tosney KW, Liegel LH: Creating effective poster presentations: AMEE Guide no. 40. Med Teach. 2009; 31(4): 319–321. PubMed Abstract | Publisher Full Text\n\nMoore LW, Augspurger P, King MO, et al.: Insights on the poster preparation and presentation process. Appl Nurs Res. 2001; 14(2): 100–104. PubMed Abstract | Publisher Full Text\n\nMoule P, Judd M, Girot E: The poster presentation: what value to the teaching and assessment of research in pre- and post-registration nursing courses? Nurse Educ Today. 1998; 18(3): 237–242. PubMed Abstract | Publisher Full Text\n\nRandviir E, llingworth S, Baker M, et al.: Dataset 1 in: Twittering About Research: A Case Study of the World’s First Twitter Poster Competition. F1000Research. 2015. Data Source\n\nRowe N, Ilic D: What impact do posters have on academic knowledge transfer? A pilot survey on author attitudes and experiences. BMC Med Educ. 2009; 9: 71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRush K, Merritt-Gray M, Noel J: The poster assignment: a connected teaching strategy for increasing student comfort with issues of sexuality. Nurse Educ Today. 1995; 15(4): 298–302. PubMed Abstract | Publisher Full Text\n\nShalom C: Established and evolving spoken research process genres: plenary lecture and poster session discussions at academic conferences. English for Specific Purposes. 1993; 12(1): 37–50. Publisher Full Text\n\nShelledy DC: How to make an effective poster. Respir Care. 2004; 49(10): 1213–1216. PubMed Abstract\n\nTaggart HM, Arslanian C: Creating an effective poster presentation. Orthop Nurs. 2000; 19(3): 47–49, 52. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10299",
"date": "21 Sep 2015",
"name": "Graham Scott",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting paper that describes and evaluates a Twitter poster conference. The authors have clearly highlighted the potential reach that Twitter provides scientists wishing to engage others with their work and the ways in which Twitter as a medium has distinct advantages over traditional face to face interaction. In doing so this paper provides a useful starting point for a discussion about the use of social media as a conference format. The authors acknowledge the limitations of twitter (e.g. limited ability to have an in depth discussion) but from the presented data they are unable to discuss this in any detail. In my opinion this is an area that should be investigated further. As an initial foray into the field this is a (very necessary) descriptive rather than investigative piece. In order that I might fully evaluate the value of the model I would have like to have more information about the broader themes of the tweets themselves – what proportion of them asked/answered a relevant question? How many were simply an indication of approval or an acknowledgement of participation in the virtual meeting etc. I don’t think that this information needs to be added to the current paper (it makes a valuable initial contribution) but I would encourage the authors to follow this work with such an analysis if that is possible. There is one minor change that I think would increase the initial readability of the paper. On first reading I found myself confused about the nature of the conference, perhaps revealing a prejudice based on prior experience and pre-conception I assumed that the Twitter poster conference was an adjunct to a physical meeting rather than a stand alone event. It may help readers to avoid this mistake if the authors explained that more clearly in the abstract?",
"responses": [
{
"c_id": "1732",
"date": "29 Dec 2015",
"name": "Craig Banks",
"role": "Author Response",
"response": "We thank the referee for their insightful comments and feel that they are of benefit to the manuscript both now and in the future.We are in agreement that this area should be investigated further, and have included some information as to how we will extend this work in the future, specifically relating to the nature of Twitter exchanges and in developing a level of meaning based upon the information within exchanges. The referee makes a valid point regarding the ambiguity surrounded this event as a standalone conference. The abstract has been changed to reflect the nature of the conference to make this less confusing."
}
]
},
{
"id": "11113",
"date": "10 Dec 2015",
"name": "Durhane Wong-Rieger",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 concept and successful execution of this \"experiment\" makes it worth publishing. The data collected supports the premise that sharing a poster through a dedicated # will attract participants. However, some of the other concerns raised about the limitations of more traditional research poster sessions do not seem to be addressed. It is not clear why this was conducted as a competition and whether there would have been the same level of participation without this element. So, the level of engagement might have been biased by a factor not usual in a poster session. It would have been very useful to have a \"qualitative\" content analysis of the tweets, or at least a sample of tweets to understand what was exchanged in the messages. This would add to understanding of the potential value of a Twitter-fest for scientific exchange. At least an analysis of the scope or \"build\" of discussion would be useful. How many exchanges were two, three or four exchanges? How many exchanges were builds on a core comment or additions to an initial exchange?It may also have been beneficial to have a discussion as to the implications of the countries participating.\n\nThe conclusions of success based on \"ease of knowledge exchange, lack of travel requirement, and reduction in carbon footprint\" are somewhat questionable, given that these are the qualities of social media (especially Twitter) and so do not reflect on the success or value of this activity.It may have been useful to solicit some feedback from participants, including the presenters, as to how they felt about the experience, the quality of the exchange, and the interest in doing again.",
"responses": [
{
"c_id": "1731",
"date": "29 Dec 2015",
"name": "Craig Banks",
"role": "Author Response",
"response": "We thank the reviewer for providing an insightful review that will not only help improve the manuscript, but will also affect our future work.While we understand the reviewer’s thought regarding the competitive element, we feel that most scientific conferences of this nature offer prizes for the best posters. With this in mind, we feel that the competitive element is no different to other conferences and assume that the participation does not change based upon this. We have noted this in the experimental section.The referee makes an excellent point regarding the nature of exchanges. We feel that with the information we have collected, we cannot extract enough quality data to report within this paper, but future work will focus more on a detailed analysis of Twitter interactions and develop a model for the level of meaning in each interaction. We have pointed this out within the updated manuscript in the conclusion, and have also discussed in the text that we feel further detail regarding exchanges would be useful. We agree that a feedback method should have been incorporated and this will be included in future works. The discussion section has been changed slightly to reflect the fact that this was an opportunity missed. We have removed the conclusion regarding lack or travel and carbon footprint, but remain optimistic that Twitter improves ease of knowledge exchange, if not directly then indirectly."
}
]
},
{
"id": "11359",
"date": "15 Dec 2015",
"name": "Michael Bales",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 academic conference and poster competition that occurred online in February 2015. Researchers were invited to share their work via Twitter in the form of an academic poster during a specified window of time on the day of the conference. The authors compare and contrast the online conference with poster sessions at traditional academic conferences, identifying several advantages and disadvantages.The article is well-written and serves as a good contribution to a growing body of work focusing on the changing modalities of scholarship and academic collaboration.Several minor comments:In the description for Table 1, the authors state that \"on average, every poster potentially received in excess of 4500 views...\" So that this caption can stand on its own, it would be helpful to clarify why the word \"potentially\" is used in this case.In Figure 2 there one line for \"Reach\" and another for \"Tweets\", but the Y-axis is labeled \"Impressions\". Given that readers may be expecting a second Y-axis, the caption for Figure 2 should be sufficient to explain these three concepts.",
"responses": [
{
"c_id": "1735",
"date": "29 Dec 2015",
"name": "Craig Banks",
"role": "Author Response",
"response": "We thank the reviewer for their comments and have endeavored to respond appropriately.The word \"potentially\" is used because we feel that there is no way that each poster had over 4200 views - the figure derives from the number of impressions divided by the total contributors, and assumes that every impression is knowingly observed. We have included this in the manuscript. The caption in Figure 2 has been defined more appropriately."
}
]
}
] | 1
|
https://f1000research.com/articles/4-798
|
https://f1000research.com/articles/5-1437/v1
|
20 Jun 16
|
{
"type": "Research Note",
"title": "Combining combing and secondary ion mass spectrometry to study DNA on chips using 13C and 15N labeling",
"authors": [
"Armelle Cabin-Flaman",
"Anne-Francoise Monnier",
"Yannick Coffinier",
"Jean-Nicolas Audinot",
"David Gibouin",
"Tom Wirtz",
"Rabah Boukherroub",
"Henri-Noël Migeon",
"Aaron Bensimon",
"Laurent Jannière",
"Camille Ripoll",
"Victor Norris",
"Armelle Cabin-Flaman",
"Anne-Francoise Monnier",
"Yannick Coffinier",
"Jean-Nicolas Audinot",
"David Gibouin",
"Tom Wirtz",
"Rabah Boukherroub",
"Henri-Noël Migeon",
"Aaron Bensimon",
"Laurent Jannière",
"Camille Ripoll"
],
"abstract": "Dynamic secondary ion mass spectrometry (D-SIMS) imaging of combed DNA – the combing, imaging by SIMS or CIS method – has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to 13C-labeling via the detection and quantification of the 13C14N- recombinant ion and the use of the 13C:12C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS.",
"keywords": [
"DNA isotope labeling",
"PCR",
"DNA extraction",
"silicon chip",
"surface coating",
"isotope recombination"
],
"content": "Introduction\n\nThe CIS method (for combing-imaging by SIMS) combines DNA combing on a silicon surface, flooding the surface with cesium, and the sensitive imaging technique of dynamic secondary ion mass spectrometry (D-SIMS) as provided by a Cameca NanoSIMS 50, a machine that allows five or seven different masses to be detected simultaneously, depending on the version1,2. This method may be of interest for those who wish to obtain fine-scale, quantitative information on DNA replication and protein-DNA interactions at the level of single molecules without the need to modify these molecules. Previously, we have used the CIS method and a NanoSIMS 50 to obtain quantitative images of single DNA fibers combed on modified, SIMS-compatible, silicon surfaces with lengths similar to those expected for the B-conformation and with a resolution of 50 nm, i.e., 150 bp3.\n\nVariations in the rates and/or directions of DNA synthesis are implicated in many processes, including gene expression4, nutrient sensing involving the alarmone (p)ppgpp5, R-loop-mediated replication6 and DNA repair7. Variations between replisomes within the same chromosome have been observed using BrdU and light microscopy8. To study such variations on the 50 nm scale, CIS might usefully be extended to image not only the 12C15N- and 13C15N- recombinant ions but also other ions. Here, we describe in detail the protocols needed for CIS, we report using CIS to image the 13C-, we suggest ways in which 13C-imaging could be improved, and we mention possible applications.\n\n\nMaterials and methods\n\nIn principle, DNA could be extracted from any organism to perform CIS. DNA generated in vitro can also be used. For the experiment reported here, for example, we extracted DNA from the 168 (trpC2) strain of Bacillus subtilis and a derivative (MT119, trpC2 leuB6 r- m-) harboring a 21.1 kb long, chloramphenicol-resistant plasmid (pHV1431 plus insert of 10.3 kb)3. On modified silicon surfaces at concentrations of 0.2 µg/mL, most of the DNA is combed as single fibers. Higher concentrations can lead to fibers running together whilst lower concentrations result in an insufficient number of fibers on the images. Combing works well with buffers containing NaCl at ionic strengths of around 0.2M.\n\nBacterial culture medium: 14g/L of K2HPO4, 6g/L of KH2PO4, 1g/L of sodium citrate, 10mM MgSO4, 0.01% (w/v) of tryptophan, 0.005% (w/v) of leucine, 0.2%(w/v) of 13C-glucose (Isotec) and 0.8g/L of 15NH4Cl (Isotec). Lysis buffer: 50mM of Tris-HCl (pH8.0), 10mM of EDTA (pH8.0), 150mM of NaCl and 5mg/ml of lysozyme. Proteinase K: Final concentration of 0.2mg/mL (Roche). Sarcosyl Buffer: Final concentration of 1.2%. Phenol/chloroform: Solution PCI: phenol-chloroform-isoamylalcohol (25:24:1 v/v) (the phenol is first saturated in NaCl 150mM and buffered at pH of about 7); Solution CI: chloroform-isoamylalcohol (24:1 v/v). RNase: Final concentration of 20µg/mL (Roche).\n\nOther materials required are: 100% cold ethanol; 70% (v/v) ethanol; NaCl solution: 0.2M to 1M; PureYieldTM plasmid midiprep system (Promega); restriction enzyme; 0.3M Potassium acetate pH7.0; TaKaRa EX TaqTM system (TaKaRa); Expand Long Template PCR System (Roche Applied Science); QIAquick PCR Purification kit (QIAGEN).\n\nDNA preparation from bacterial cultures. DNA can be prepared from any cells using a procedure that removes contaminating peptide and RNA molecules. In the case of the labeling and extraction of B. subtilis DNA, B. subtilis cells were cultivated at 30–37°C for ~20 generations in bacterial culture medium containing stable isotopes (here, 13C and 15N, see above) to saturation. Then, to prepare chromosomal DNA free of peptides and RNA, one milliliter of a freshly saturated culture was centrifuged at 15000 g for 2 min at 4°C. The supernatant was discarded. Pelleted cells were resuspended in 0.5 mL of lysis buffer. The cell resuspension was incubated for 20 minutes at 37°C. Proteinase K was added to a final concentration of 0.2 mg/mL in addition to 20µL of sarcosyl buffer 30% (1.2%, final concentration) and incubated for 20 min at 65°C. To remove peptides and cell fragments by a phenol/chloroform treatment, we lowered the temperature of the sample on ice for 3 min and then added 500µL of solution PCI (see Preparation of DNA) at 4°C and vortexed strongly for 30 s. The mixture was centrifuged at 15000 g for 15 min at room temperature in a benchtop centrifuge. The aqueous solution was recovered and 500 µL of solution CI was added to it (see Preparation of DNA). This mixture was vortexed strongly and centrifuged as above. RNase was added to a final concentration of 20 µg/mL to the aqueous, nucleic acid-containing phase and incubated for 10 min at 37°C. DNA was purified by a second phenol/chloroform extraction as above. We added 2.2 volume of 100% -20°C ethanol to the aqueous solution and centrifuged at 15000 g for 20 min at 4°C in a benchtop centrifuge. The supernatant was discarded and the DNA pellet was washed in 70% cold ethanol. This mixture was centrifuged again for 10 min at 4°C at 15000 g and the supernatant was discarded. The DNA pellet was dried for 10 min under vacuum and resuspended carefully in pure water for at least 12 h at room temperature (note that incomplete solubilization of ethanol-precipitated DNA severely perturbs combing). DNA concentration was measured by absorbance at 260/280 nm using a Nanodrop 2000 (Thermo Scientific). Depending on the fragment length, DNA concentration should be adjusted to within the range 0.2 to 2 µg/mL by dilution in NaCl solutions that may range up to to 1M; here, we used DNA at 0.2µg/mL in 0.2M NaCl.\n\nIn this paper, we only report CIS in the case of chromosomal DNA. CIS can also be used to analyse plasmid DNA (not shown). In the case, for example, of a 20 kb plasmid from B. subtilis, plasmid DNA may be extracted using the PureYieldTM plasmid midiprep system (Promega Corporation, Madison, WI, USA), followed by linearization of the plasmid by a single cutter restriction enzyme according to suppliers’ instructions (e.g. New England BioLabs, Inc., Hitchin, UK) to give fragments of the appropriate size; this linearization is needed before combing because of the difficulty of combing supercoiled circular molecules. After linearization, the DNA can be purified by the phenol/chloroform procedure and recovered by ethanol precipitation in the presence of 0.3 M potassium acetate pH 7.\n\nDNA preparation by PCR (protocol used to prepare PCR-generated DNA fragments 1-20 kb long). DNA fragments generated by PCR can be analyzed by CIS3. We routinely produce fragments <5 kb using the TaKaRa Ex TaqTM system (Takara Shuzo Co., Ltd, Shiga, Japan). For products of 5-20 kb, we use the Expand Long Template PCR System from Roche Applied Science (Mannheim, Germany). The PCR products can be purified with the QIAquick PCR Purification kit (QIAGEN GmbH, Hilden, Germany). PCR products can then be diluted for CIS as described above.\n\nThis experiment required an efficient fume hood. The materials used were: P-type 100 crystalline silicon wafers (Siltronix) with a resistivity > 1 Ω cm (typically 10 mm × 10 mm × 380 μm); Piranha solution obtained by mixing 3 volumes of 96% of sulfuric acid [VLSI (very large-scale integration)-grade] and 1 volume of 30% hydrogen peroxide (VLSI-grade), to be used immediately; 50% hydrofluoric acid (VLSI-grade); acetone; isopropyl alcohol (iPrOH); chloroform (CHCl3); ethanol (EtOH); 1-tetradecene; a photochemical reactor for UV irradiation (λ=312 nm) (our home-made reactor has 8 tubes in a circle around a Schlenk tube with a fan at the bottom to limit any increase in temperature); a goniometer system (DIGIDROP, GBX, France).\n\nPreparation of hydrogenated-terminated silicon surfaces. In the following preparation, it should be noted that the piranha solution is a powerful oxidant that reacts violently with organic materials; it can cause serious skin burns and must be handled with great care in a well-ventilated fume hood while wearing appropriate chemical safety protection. Moreover, HF is a hazardous acid that can cause severe damage to tissues if burns are not treated properly. Etching of silicon should therefore be carried out in a fume hood with the right safety measures, which include a face shield and double-layered nitrile gloves.\n\nThe silicon surface was cleaned in an ultrasonic bath for 5 min periods in acetone and isopropylic alcohol. It was then rinsed extensively with ultrapure water and immersed for 20 min in a piranha solution to remove organic contaminants on the surface. This was followed by the immersion of the clean surface in an aqueous solution of HF (50%, as provided by the supplier) to generate a hydrogen-terminated surface (Si-H)9. This surface was washed extensively with water and then dried by blowing with nitrogen. The resulting surface typically had a contact angle of 85° for a 1 µL water droplet. Note that freshly prepared Si-H surfaces were used immediately for DNA combing or for grafting hydrocarbon chains (see below). Before combing, coated silicon surfaces were protected from dust and dried because a thin film of water greatly reduces DNA adsorption.\n\nFormation of organic monolayers on hydrogen-terminated silicon surfaces. To obtain highly hydrophobic surfaces, alkenes with C14 alkyl chains were grafted onto freshly prepared Si-H surfaces using the hydrosilylation reaction of 1-tetradecene with the hydrogen-terminated silicon surface9. The freshly hydrogen-terminated silicon surface (see above) was immersed in a Schlenk tube containing 10 ml of previously deoxygenated, neat 1-tetradecene under nitrogen bubbling. This was then irradiated at 312 nm in a photochemical reactor for 3 h. We removed unreacted and physisorbed 1-tetradecene by rinsing with chloroform and ethanol at room temperature. The silicon substrate was dried under a stream of nitrogen. We verified that water contact angles (using deionized water in the ambient atmosphere at room temperature) are around 104° as measured to an accuracy of ± 2° with a remote, computer-controlled, goniometer system (N.B., protect the surface from dust, see above).\n\nCombing on silicon. The 'drop' and 'lift' methods10,11 can both be used to comb DNA on the Si-C14 surface. Both 'drop' and 'lift' methods entail the DNA becoming attached to the surface and then drawn out and aligned perpendicular to the triple line or the meniscus, respectively. The ‘drop’ method entails depositing 10 µL of prepared DNA solution on the silicon surface, incubating it for 10 min on the bench, tilting the surface with tweezers to 45° to cause the drop to roll off the surface, and washing the surface by immersion in water and rapidly air-drying; the quality of combing via this method can be adversely affected by vibrations occurring during the movement of the drop. Here, we used the ‘lift’ method, which entails immersing a part of the silicon surface in a DNA solution (see above); after 5 min incubation, the silicon surface was pulled out of the solution at a constant speed (600 µm/min) and rapidly air-dried. In both ‘drop’ and ‘lift’ methods, washing the surfaces after combing helps avoid perturbation of D-SIMS analysis by saline crystals. The tightly controlled, motor-driven ‘lift’ method gives more reproducible, better quality results but takes longer and requires more DNA than the manual ‘drop’ method. The ‘lift’ method is better than the drop method for fragments less than 5 kb; the ‘drop’ method is satisfactory with fragments equal to or greater than 5 kb.\n\nCs flooding method. To avoid a premature destruction of thin samples before SIMS detection12, a cesium flooding system is recommended to deposit neutral cesium on samples13,14. We used a UHV Cs evaporation system which is available for purchase from the Luxembourg Institute of Science and Technology (LIST) . It comprises a neutral cesium evaporator and an independent stand-alone UHV chamber. A suitcase under UHV (10-8 to 10-10 Torr) was used to transfer samples to the NanoSIMS50 thereby avoiding an immediate reaction between the neutral cesium deposit and air. To analyze combed DNA (as performed here), we flooded the surface with Cs0 at 1.5 Å/s for 1800 s before SIMS imaging. An alternative to cesium flooding is to coat samples with Au; a Cressington Sputter Coater can be used to deposit a layer of Au, approximately 60 nm thick, on wafers with combed DNA but the quality of the images subsequently obtained is often poor.\n\nNanoSIMS 50 analyses. In SIMS analyses of samples labeled with carbon and nitrogen, there are several factors that should be borne in mind. First, carbon can be detected as C- and in multi-clusters that include CN- and C2- (N.B. CN is easier to ionize and therefore detect than C and C2 because the electron affinity is 3.8eV for CN-, 1.26eV for C- and 3.27eV for C2-). Second, nitrogen can be detected in the multi-cluster CN (note that nitrogen forms fewer types of multi-clusters thant carbon even though it can form to a very limited degree multi-clusters that include NO, N2H, and NS). Third, the probability of formation of the CN multi-cluster in the mixing-recombination under the primary beam is related to the distance between the carbon and nitrogen atoms15; this means that differentially labeled macromolecules can be colocalized if they are within 2 nm of one another; it also means that the carbon and nitrogen in the DNA are more likely to recombine with one another than with carbon and nitrogen that are further away. Note that even a trace amount of contaminants is an important problem for the highly sensitive CIS method and strenuous efforts should therefore be taken to protect samples from contamination by, for example, an atmosphere containing C and N. Silicon surfaces with combed DNA were analyzed in the multi-collection image mode of the NanoSIMS 50 (Cameca, Gennevilliers, France). The NanoSIMS 50 was used in the negative secondary-ion mode with the Cs+ primary ion beam. In this case high spatial resolution images were obtained using a primary beam around 0.5–1.0 pA in intensity and an impact energy of 16 keV. The surface was rastered by the cesium beam on a surface from 5 × 5 to 15 × 15 µm2. The instrument was tuned to limit the dispersion in aperture and energy in order to obtain a minimal mass resolution of M/ΔM = 5000 (10% height peak measurement).\n\nAphelion 3.2 was used to analyze the results pixel by pixel or line by line, whilst ImageJ plus the OPEN MIMS plug-in (which opens the .im format16 and is available from http://www.nrims.harvard.edu) was used to analyze the data in an ROI (Region Of Interest). The data were summed and the images colored using WinImage 2. The images of the 12C (m = 12.000000), and/or 13C (m = 13.00335484), 12C14N (m = 26.003074), 12C15N (m = 27.00010898) or 13C14N (m = 27.00642885), 13C15N (m = 28.00346382) were acquired simultaneously in 256 × 256 pixels with a dwell time of 10 ms per pixel. Note that 12C15N and 13C14N cannot be detected simultaneously in the same ‘sputter section’. The sulfur 32S (m = 31.9720718) distribution can be also acquired to control the quality of the preparation.\n\n\nResults\n\nThe CIS technique reveals individual DNA fibers of a wide variety of lengths lying in parallel (Figure 1). These fibers are particularly clear in the 13C15N image of the DNA (Figure 1a) but, importantly, they can also be discerned in the 13C14N image (Figure 1b). The images differ because (1) the coating on the silicon surface contains a large amount of the natural isotopes of carbon of which 1% is 13C; this 1% can then recombine with contaminant nitrogen to generate a high background of 13C14N that partially masks the signal from the labeled DNA (even though this DNA is 99% 13C) and (2) the background of 13C15N is low since this ion arises at a frequency of 1:25000 in combinations of naturally occurring carbon and nitrogen).\n\nThe DNA was combed at 0.2 μg/mL on a Si_C14 wafer using the lift method, the wafer surface was covered with cesium and analyzed with a NanoSIMS 50. Primary beam intensity, 1 pA; dwell time, 30 ms; field of view, 15 μm × 15 μm; 256 × 256 pixels; scale bar = 1 μm. (a) 13C15N, (b) 13C14N, (c) Isotope ratio 13C/12C, and (d) 13C15N+13C14N. (a) and (b) are the results of adding the counts from three successive sputter sections, (c) is the ratio between the two sets (13C and 12C) of three successive sputter sections, and (d) is the three sputter sections of (a) plus the three of (b). The count numbers of (a), (b) and (d), and the count ratio of (c) are given on the linear color scales.\n\nMost of the fibers have similar counts throughout their lengths but, in a couple of cases, two fibers overlap and this results in more 13C15N- recombinant ions being detected (the red stretches in Figure 1a). The relative constancy of the counts and the similarity between the calculated lengths of the DNA and the lengths estimated by SIMS support the claim that CIS does not stretch or condense DNA3. However, an exception to this may occur when the DNA is so short that it cannot be combed properly, as is probably the case of the red spots of a few nm (Figure 1a).\n\nThe fibers in the image of the isotope ratio 13C/12C image (Figure 1c) are difficult to detect in their entirety. One reason for this difficulty is that the natural carbon in the coating on the surface contains 13C, as mentioned above. A second reason is that the yield of carbon in the C- form is relatively low and therefore the ratio can fluctuate (see Materials and methods). A third reason is that the carbon in the DNA reacts with neighboring atoms in the DNA (and elsewhere) such as nitrogen – and indeed carbon itself – to give multi-clusters (see Materials and methods); this means that much of the 13C is being distributed into multi-clusters that include 13C15N, 13C14N, 12C13C and 13C13C. It is therefore significant that, even in these unfavorable conditions for detecting 13C via the 13C:12C ratio, 13C-labeled fibers can just about be distinguished (Figure 1c).\n\nIn the experiment reported here, 98% of the nitrogen in the DNA is 15N. This means that the counts of 13C14N coming from recombination between the atoms within the DNA are nearly 98% lower than they would be if all the nitrogen in the DNA were present as the 14N isotope. To answer the question of whether labeling with 13C alone (i.e., without 15N-labeling) is sufficient to allow DNA to be detected, we added the counts of 13C15N (Figure 1a) to those of 13C14N (Figure 1b) to obtain an image of 13CN (Figure 1d). The fibers are again clear, showing that 13C labeling is indeed sufficient to allow DNA to be detected.\n\n\nDiscussion\n\nPreviously, we have suggested that CIS could be used in conjunction with pulse-labeling with different isotopes to identify origins of replication or to study local variations in the rate of DNA elongation resulting from signals generated inside or outside cells or from addition of drugs3. Such pulse-labeling can be readily based on 15N since this is relatively rare naturally (0.36%) and enriching in 15N therefore makes fibers readily detectable. Here, we ask whether 13C labeling with 13C alone might suffice for detection via CIS. We try to answer this question in the context of having labeled the fibers with both 13C and 15N (Figure 1a). Even in these conditions, the image of the 13C14N- recombinant ion does allow fibers to be discerned, albeit with difficulty (Figure 1b). This difficulty is due not only to reduction of the ratio by surface contamination by carbon (which contains 1% 13C) but also to the recombination that generates multi-clusters (see Materials and methods); the latter includes recombination between the 13C and 15N in the fibers to give 13C15N (it is therefore not surprising that the fibers are hard to detect in Figure 1c using the ratio of the 13C- and 12C- ions). Another way to decide whether 13C14N can be used for imaging when DNA is labeled with just 13C is to add the counts from both 13C14N and 13C15N by combining the results in Figure 1a & 1b so as to increase the counts of 13C (Figure 1d). This shows that the fibers can indeed be discerned (although it should be noted that here both strands are labeled rather than just one strand being labeled as would occur after a short pulse). This result is significant because it means that, in the same experiment, one could label the DNA successively: first with 13C in 14N medium to detect 13C14N and then with 15N addition to detect 13C15N. One might even succeed in detecting three consecutive sequences, for example, by (1) labeling with 13C (via U-13C-Glc) to detect the 12C13C- or 13C13C- recombinant ion (or indeed simply the 12C- and 13C- ions), then by (2) labeling with 15N (via 15NH4Cl) to detect 13C15N, and finally by (3) replacing either the 13C with 12C (via U-12C-Glc) to detect 12C15N or the 15N with 14N (via 14NH4Cl) to detect 13C14N. Such consecutive labeling would allow investigation of local variations in the velocities of replication.\n\nIn principle, CIS could permit testing of several hypotheses. These include (1) the idea that the elongation step of DNA replication is coupled to central carbon metabolism17–19 and that this changes the local structure of the DNA20,21 or changes the relative copy numbers of genes4 and thereby affects the phenotype, (2) the idea that the strand separation resulting from ion decondensation is responsible for the initiation of DNA replication22, (3) the idea that the time to rereplicate the chromosomes in a cell population growing in steady state is highly variable since these populations are phenotypically diverse23,24, (4) the idea that the replication of a particular species of bacteriophages or viruses is very diverse. CIS could also be used to study the fine-scale interaction between DNA sequences and RNA, proteins and polyamines. Finally, CIS might eventually be used to study the modification of individual DNA fibers due to the covalent addition of methyl and acetyl groups, sugars, and other molecules, always providing that these molecules could either be labeled specifically by very rare isotopes such as 14C or bound specifically by antibodies or aptamers that were themselves labeled.\n\nThe value of such potential applications of CIS depends on the number of different ions that can be detected and on the quality of this detection (which depends on factors such as the signal:background ratios, the dwell time, the intensity of the primary beam, and the use of serial sputter sections). With the latest NanoSIMS 50L version of the NanoSIMS range, seven different masses can be detected simultaneously in the acquisition of a single ‘sputter scan’25, which increases the numbers of carbon multi-clusters that can be analyzed. Finally, the quality of detection of 13C- and the recombinant 13CN- ions could be improved by coating the silicon surface with carbon depleted in 13C. In principle, therefore, CIS could distinguish between DNA labeled so as to give consecutive stretches enriched in (1) 13C (detectable as 13C13C), then (2) 13C and 15N (detectable as 13C15N) and, finally, (3) 12C (detectable as 12C15N) (note there are other possible combinations). CIS could, of course, be extended further still if the DNA were labeled with Br or I or even 14C26.\n\n\nConclusion\n\nCIS combines the advantages of DNA combing and SIMS in providing high sensitivity and high resolution since DNA fragments down to 1500 nm can be imaged at a resolution of 50 nm. Moreover, the labeling with stable isotopes does not significantly perturb cells. The fact that CIS can detect 13C14N increases the number of separate labels available to the technique, thereby making CIS particularly valuable for studying phenotypically important variations in the elongation of DNA replication, as well as the processes of initiation of replication, recombination and repair. The increase in the repertoire of labels available to CIS also supports the case for it being adapted to study the interaction between DNA and other molecules, including proteins and drugs, and perhaps even to the precise localization of covalent modifications to DNA.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for Combing-Imaging by SIMS of bacterial DNA, 10.5256/f1000research.8361.d12027827",
"appendix": "Author contributions\n\n\n\nVN had the initial idea. AC, AFM, YC, JNA, DG, TW and LJ performed experiments. All authors participated in the data analysis and the writing of the paper.\n\n\nCompeting interests\n\n\n\nThe Luxembourg Institute of Science and Technology markets cesium-flooding equipment. Genomic Vision offers a DNA-combing service.\n\n\nGrant information\n\nThis work was partly supported by the FP7 EU Network of Excellence, Nanobeams n°500440 to Henri-Noël Migeon.\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 Stephane Alexandre for help with Atomic Force Microscopy.\n\n\nReferences\n\nGuerquin-Kern JL, Wu TD, Quintana C, et al.: Progress in analytical imaging of the cell by dynamic secondary ion mass spectrometry (SIMS microscopy). Biochim Biophys Acta. 2005; 1724(3): 228–238. 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}
|
[
{
"id": "14618",
"date": "27 Jun 2016",
"name": "Bruno Fabre",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 significant insights in a novel SIMS imaging technique focusing on the imaging of DNA fibers immobilized on modified silicon surfaces. The conclusions are adequately supported by the experimental data. Consequently, I recommend indexation of this as is.",
"responses": []
},
{
"id": "14893",
"date": "11 Jul 2016",
"name": "Freddy C. Adams",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a well-written report on work concerning the use of the Cameca NanoSims50 instrument for imaging of DNA fibers immobilized on silicon substrates. The work is a follow-up of earlier papers on the same subject by the present consortium. It contains work that is original and of interest for the scientific community. It shows the value of potential applications of Combing-Imaging with SIMS (CIS) and leads the way to further work with this ultra-sensitive chemical imaging tool. I fully support its indexations.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1437
|
https://f1000research.com/articles/5-1436/v1
|
20 Jun 16
|
{
"type": "Review",
"title": "Recent advances in understanding and treating vasculitis",
"authors": [
"Matthew J. Koster",
"Kenneth J. Warrington",
"Kenneth J. Warrington"
],
"abstract": "Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) are near universally fatal conditions if untreated. Although effective therapeutic options are available for these diseases, treatment regimens are associated with both short- and long-term adverse effects. The recent identification of effective B-cell-targeted therapy with an anti-CD20 monoclonal antibody has transformed the treatment landscape of AAV. Questions, nevertheless, remain regarding the appropriate timing, dose, frequency, duration, and long-term effects of treatment. The aim of this article is to provide an overview of the current information, recent advances, ongoing clinical trials, and future treatment possibilities in AAV.",
"keywords": [
"systemic vasculitides",
"ANCA-associated vasculitides",
"vasculitis",
"B-cell"
],
"content": "Introduction\n\nThe systemic vasculitides are a heterogeneous group of diseases characterized by inflammation of blood vessels, resulting in tissue damage and end-organ dysfunction. Although relatively rare, if left untreated, these conditions lead to significant morbidity and mortality. While the etiology of the primary vasculitides is still unknown, a better understanding of their pathogenic mechanisms in combination with an expanded armamentarium of targeted therapeutics has led to significant advances in treatment. Among the primary systemic vasculitides, the treatment of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) has most notably benefited from these recent advances. Landmark studies in AAV showing the efficacy of B cell depletion have led to rapid clinical utilization of rituximab (RTX), an anti-CD20 monoclonal antibody which targets and depletes premature and mature B lymphocytes. However, once patients are in remission, there is controversy surrounding the appropriate timing, dose, frequency, duration, and long-term effects of this treatment. The purpose of this review is to provide an overview of the current information, recent advances, ongoing clinical trials, and future treatment possibilities in AAV.\n\n\nInduction of remission\n\nThe AAVs comprise granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). These multisystem, immune-mediated conditions primarily affect small blood vessels, most commonly small arteries, arterioles, capillaries, and venules of the lung and kidney. For patients with severe systemic AAV, the mainstay of remission induction treatment has included high-dose glucocorticoids and oral or intravenous (IV) pulse cyclophosphamide (CYC).\n\nAlthough effective in 70–90% of patients with AAV1, the use of CYC, particularly in refractory and relapsing disease, is limited owing to cumulative toxicity and serious adverse events which can include infertility, bladder hemorrhage, severe cytopenias, serious infection, and increased risk of malignancy. Because of these associated side effects, reducing the overall cumulative exposure to CYC during remission induction has been advocated. De Groot and colleagues demonstrated that pulse dose CYC induced remission of AAV as well as daily oral regimens while reducing the cumulative CYC dose by half and significantly decreasing the frequency of treatment-related leukopenia2. Despite limited power to determine the effect of the evaluated induction regimens on relapse, longer-term follow-up identified that patients receiving pulse dose CYC had a twofold increased risk of relapse compared to those receiving daily oral CYC3. This was particularly pronounced among those with anti-proteinase 3 antibodies (PR3-ANCA). As such, limiting CYC treatment may not be as beneficial for some patient subsets.\n\nIn AAV, CYC appears to exert a greater effect on B cells compared to T cells4. B cells and their progeny appear to be central in the pathogenesis of AAV through the production of ANCA, as well as T cell co-stimulation and cytokine production5. Furthermore, the number of circulating activated B cells correlates with disease activity6. The identification of these pathogenic roles provided the framework for two landmark randomized trials to evaluate B-cell-targeted therapy for remission induction in patients with GPA and MPA.\n\nThe Rituximab vs. Cyclophosphamide in ANCA-associated Renal Vasculitis (RITUXVAS) trial included 44 patients with newly diagnosed GPA or MPA and renal involvement. In addition to receiving high-dose glucocorticoids, patients were randomized (3:1) to induction treatment with either RTX (375 mg/m2) weekly for 4 weeks with two IV pulses of CYC or IV CYC pulses for 3–6 months followed by azathioprine (AZA) maintenance7. Sustained remissions at 12 months were similar between the RTX (76%) and CYC/AZA (82%) groups, demonstrating the efficacy of RTX for remission induction in patients with AAV and significant renal dysfunction. Surprisingly, no difference in adverse events or mortality was observed. Extended trial follow-up data at 24 months8 showed that the RTX-based induction without maintenance therapy had similar longer-term outcomes to the CYC/AZA regimen with regard to relapse (21% vs. 18%) and mortality (18% vs. 27%).\n\nThe Rituximab vs. Cyclophosphamide for ANCA-associated Vasculitis (RAVE) trial was a randomized, double-blind, double-dummy, placebo-controlled, non-inferiority trial evaluating a total of 197 patients with GPA or MPA9. In contrast to RITUXVAS, RAVE included both newly diagnosed and relapsing patients comparing RTX 375 mg/m2 weekly for 4 weeks to 2 mg/kg oral CYC for 3–6 months followed by AZA maintenance. All trial participants received high-dose glucocorticoids, which were tapered off according to a standardized protocol. The RAVE trial concluded that RTX was non-inferior to CYC/AZA for inducing remission at 6 months, with 64% of RTX patients and 53% of CYC/AZA patients reaching this endpoint. However, for GPA/MPA patients entering the study with relapsing disease, RTX appeared more efficacious with 67% (34/51) of patients reaching remission at 6 months compared to 42% (21/50) with CYC/AZA. Extended follow-up results showed similar long-term efficacy between the two regimens with 48% and 39% of patients in sustained remission at 12 and 18 months in the RTX arm and 39% and 33% in sustained remission at 12 and 18 months in the CYC/AZA arm, respectively10.\n\nThough it was anticipated that induction with RTX might be safer than CYC, it is of note that the number of adverse events in both the RITUXVAS and RAVE studies was similar between the two treatment arms7,9. Potential reasons may include identification of treatment-related complications due to concomitant high-dose glucocorticoids and short duration of follow-up preventing an observed difference in known long-term CYC-associated adverse events.\n\nTaken together, the results of RITUXVAS and RAVE demonstrate RTX to be equivalent to CYC for remission induction of AAV among treatment-naïve patients and likely superior to CYC for relapsing disease.\n\n\nRemission maintenance\n\nGPA and MPA, however, are chronic diseases, and up to 40–50% of patients can have a relapsing course despite immunosuppressive treatment. Traditionally, patients treated with a CYC induction regimen are transitioned to an oral conventional immunosuppressive agent, while glucocorticoids are gradually tapered. Trial data from the Cyclophosphamide vs. Azathioprine for Early Remission Phase of Vasculitis (CYCAZAREM) study found that early substitution with AZA following a minimum of 3 months of oral CYC therapy had similar overall outcomes to a 12-month oral CYC regimen1. However, a slight numerical increase in relapse was observed in longer-term follow-up among those receiving shorter CYC courses11. Further research is needed to determine if certain subsets of patients would benefit from a duration of CYC treatment tailored to their risk of relapse and likelihood of treatment-related morbidity11. While AZA is the most commonly used immunosuppressive agent for remission maintenance, methotrexate has also shown similar efficacy12, provided patients have adequate renal function. Mycophenolate mofetil, on the other hand, has been shown to be inferior to AZA for remission maintenance but remains an alternative for patients intolerant of other immunosuppressive options13. In addition to conventional oral immunosuppressive medications, repeated administration of RTX may also be a viable option for remission maintenance.\n\nThe long-term follow-up data from the RITUXVAS and RAVE studies confirm that a single course of RTX is beneficial for induction. Unfortunately, relapse will occur in the majority of patients if they are not provided with maintenance treatment. This appears particularly true among those with upper respiratory tract disease, granulomatous infiltrates, or PR3-ANCA positivity14–16. Several open-label studies have evaluated the use of RTX to prevent relapse; unfortunately, heterogeneity in the timing, dose, and duration of treatment has prevented consensus on a preferred maintenance regimen. Smith et al. retrospectively studied relapsing GPA/MPA patients induced with RTX and then either observed until relapse or treated with fixed-interval RTX maintenance dosing of 1 g every 6 months17. Over 2 years, 73% of the observed group had relapsed compared to only 12% receiving fixed-interval doses of RTX. Additional investigators have observed similar efficacy of pre-emptive dosing for reducing relapse rates and prolonging relapse-free survival16,18–21, leading many clinicians to employ such treatment approaches.\n\nSeveral prospective randomized trials are currently aimed at understanding the utility of RTX as maintenance therapy (Table 1). MAINRITSAN (Maintenance of Remission using Rituximab in Systemic ANCA-associated vasculitis) is the first randomized trial to compare RTX and AZA in the maintenance of AAV22. This open-label study randomized 115 patients in complete remission following standard induction with CYC and glucocorticoids to receive either RTX (500 mg on day 1 and 14, then at months 6, 12, and 18) or AZA (2 mg/kg/day for 12 months, 1.5 mg/kg/day for 6 months, then 1.0 mg/kg/day for last 4 months). At month 28, major relapse occurred in 29% (17/58) of patients receiving AZA maintenance compared to only 5% (3/57) with RTX. Although this study demonstrates RTX may be superior to AZA in maintaining remission at 2 years, it should be noted that the AZA dose was reduced starting (already) at 12 months, a schedule not frequently employed in clinical practice. Indeed, 41% of the relapses in the AZA group occurred after treatment cessation. Therefore, it is unknown if the difference in relapse rates would have been less striking if higher doses of AZA were maintained throughout the entire study period. In order to address this question, an international collaborative trial (RITAZAREM: Clinicaltrials.gov identifier NCT01697267) is ongoing. RITAZAREM will evaluate relapsing AAV patients randomized to either RTX 1 g every 4 months for five doses or AZA 2 mg/kg/day for 24 months, more closely paralleling typical clinical practice.\n\nAZA, azathioprine; BID, twice daily; BVAS/WG, Birmingham Vasculitis Activity Score for Wegener’s Granulomatosis; Comp, comparator arm; CYC cyclophosphamide; D, day; ESRD, end-stage renal disease; Exp, experimental arm; GC, glucocorticoids; M, month; PLEX, plasma exchange; RTX, rituximab;\n\nAlthough considered to be a potentially safer alternative to CYC, the delayed adverse effects of long-term RTX maintenance in AAV are unknown. While some studies show that repeat dosing is well tolerated19,21, others demonstrate notably increased adverse events, including serious infection, late-onset neutropenia, and hypogammaglobulinemia20,23. A tailored approach to guide pre-emptive treatment decisions based on serial B lymphocyte and ANCA titer monitoring has been suggested by Cartin-Ceba and colleagues19. However, other investigators have demonstrated that these parameters are not uniformly consistent in relapse prediction17,18,24. MAINRITSAN 2 (ClinicalTrials.gov identifier NCT01731561) was organized to assess this critical knowledge gap and has been designed to compare two treatment regimens for RTX maintenance; 500 mg every 6 months vs. 500 mg whenever CD19+ lymphocytes re-populate or when ANCA titers become positive or rise. Extended follow-up of this trial with an additional 18 months of fixed dosing RTX at 6-month intervals vs. placebo is also planned (MAINRITSAN 3: ClinicalTrials.gov identifier NCT02433522). These two trials will greatly advance the understanding of RTX use in remission maintenance, and results are expected in the next 3–5 years.\n\n\nAdjunct plasma exchange\n\nThe overall benefit of plasma exchange (PLEX) in severe AAV remains uncertain. Initial evidence of efficacy with PLEX was seen in a small randomized controlled trial by Pusey and colleagues evaluating patients with anti-GBM-negative renal vasculitis25. In this trial, dialysis-independent patients improved with either standard drug therapy with adjunctive PLEX or drug therapy alone, regardless of initial serum creatinine. However, patients who were dialysis dependent at study entry experienced greater likelihood of renal recovery with the addition of PLEX compared to standard drug therapy. In a larger trial, Jayne et al.26 evaluated the effect of adjunctive therapy with PLEX or IV methylprednisolone (MP) in 137 patients with severe renal vasculitis (serum creatinine >5.8 mg/dL or dialysis dependent) receiving standard treatment with oral CYC and oral prednisone. Those randomized to PLEX had a higher rate of renal recovery and independence from dialysis compared to those treated with IV MP at both 3 months (69% vs. 49%, respectively) and 12 months (43% vs. 19%, respectively).\n\nAlthough short-term outcomes were encouraging, long-term benefits are unclear, as subsequent follow-up of this cohort for a median of 3.95 years did not identify a significant difference between PLEX and IV MP among the outcomes of end-stage renal disease (ESRD) or mortality27. A meta-analysis comprising 387 patients with AAV from nine randomized trials found that patients treated with PLEX had a significant reduction in dialysis dependence (relative risk [RR] 0.64; 95% confidence interval [CI]: 0.47–0.88) but not mortality (RR 1.01; 95% CI: 0.71–1.43)28. However, the authors note that the cumulative evidence was inadequate to conclude that PLEX effectively decreases ESRD because more than 1478 patients would have been needed to determine a 25% RR reduction with appropriate confidence28.\n\nIn addition to its use for severe renal vasculitis, PLEX has been considered for the treatment of AAV with pulmonary complications from diffuse alveolar hemorrhage (DAH). The use of PLEX in DAH, however, is limited to uncontrolled observational studies and results are conflicting. Klemmer and colleagues29 found that prompt initiation of PLEX in addition to standard induction therapy for AAV led to resolution of DAH in all 20 patients retrospectively reviewed. Resolution occurred after an average of six treatments, and no treatment-associated complications were seen. Nevertheless, the absence of a control group prevents concluding whether PLEX itself led to such improvements. In a recent retrospective study, Cartin-Ceba et al. evaluated the outcomes of 73 patients with AAV and DAH and did not observe a benefit in achieving complete remission at 6 months between adjunctive PLEX and standard induction therapy (odds ratio [OR] 0.49; 95% CI: 0.12–1.95)30. The authors further evaluated 11 uncontrolled studies, including their own, and found resolution of DAH and survival to hospital discharge was achieved in 66% (69/104) of patients treated with PLEX compared to 75% (51/68) without, tempering the likelihood that PLEX is necessary in this population30.\n\nTo address these uncertain roles of PLEX, a randomized controlled trial recruiting patients with severe renal vasculitis and/or diffuse alveolar hemorrhage secondary to AAV has been developed and is underway (PEXIVAS, ClinicalTrials.gov identifier NCT00987389). Currently, the precise role of PLEX as adjunctive therapy for remission induction in AAV remains unclear. However, given the severity of disease, it is still considered by some experts to be a reasonable option for patients with severe renal disease or dialysis dependence at the time of diagnosis. The results of PEXIVAS are highly awaited to provide further insight and recommendations.\n\n\nFuture therapeutic investigations\n\nUntil this past decade, CYC was considered the only reliable induction agent for AAV. The success of non-selective B cell depletion with RTX in AAV has opened the door for the next generation of targeted therapies focusing on the innate and adaptive immune system (Table 1).\n\nB-cell-activating factor (BAFF), also known as B lymphocyte stimulator (BLyS), appears to have a key role in the stimulation of B cell proliferation and the promotion of immature B cell survival. Increased BAFF levels favor the selection of autoreactive B cells, leading to autoantibody production31, and have been associated with autoimmune conditions such as systemic lupus erythematosus32. Elevated BAFF levels have also been seen in patients with GPA, most notably among active untreated patients33, and ANCA-stimulated neutrophils promote B cell survival through the release of BAFF34. Interestingly, researchers have observed BAFF levels increasing after B cell depletion with RTX in AAV models34. This finding demonstrates that BAFF may be an integral factor in the survival of autoreactive B cells and likely facilitates disease chronicity and relapse.\n\nBelimumab is a monoclonal antibody directed against BAFF that has been approved for the treatment of systemic lupus erythematosus. This targeted BAFF inhibitor is currently being investigated in a phase III multicenter randomized trial evaluating the efficacy and safety of this medication in combination with AZA for the maintenance of remission in GPA and MPA (BREVAS: ClinicalTrials.gov identifier NCT01663623). Additional B cell survival factors are also under investigation in AAV. Blisibimod, a fusion protein that binds both soluble and membrane-bound BAFF, is currently being considered for a phase II trial for remission induction in non-severe AAV patients receiving concomitant methotrexate.\n\nAlthough B cell dysregulation has gained center stage in the treatment of AAV, abnormal circulating and lesional T cell activation may also play a role in pathogenesis35. An open-label study evaluating abatacept, a fusion protein that blocks the co-stimulatory signal needed for T cell activation, showed disease improvement in 90% (18/20) and steroid discontinuation in 73% (11/15) of patients with non-severe AAV. However, 30% (6/20) had to terminate the study owing to increases in disease activity36, portending the likelihood that T cell activation alone is unlikely to be the critical pathway for disease control. A multicenter, phase III, double-blind, placebo-controlled trial further evaluating the use of abatacept in relapsing non-severe AAV is ongoing (ABROGATE: ClinicalTrials.gov identifier NCT02108860).\n\nGusperimus, a synthetic immunosuppressive drug derived from the antitumor antibiotic spergualin, also modulates lymphocyte function in addition to other poorly understood pathways and is a candidate drug for use in AAV37. Two open-label studies have noted disease improvement among 70–95% of patients with refractory GPA38,39. Follow-up data have also demonstrated continued efficacy and minimal toxicity with up to 5 years of treatment40,41. A clinical trial comparing gusperimus to conventional therapy is currently being designed.\n\nComplement activation, particularly component C5a, has also been recently implicated in the pathogenesis of AAV. C5a is a potent inflammatory mediator as well as a strong chemoattractant and neutrophil activator42. C5a acts as a priming agent for neutrophils, resulting in increased surface expression of the auto-antigens proteinase 3 (PR3) and myeloperoxidase (MPO). These auto-antigens interact with ANCA and subsequently stimulate the release of factors that activate the alternative complement pathway, further perpetuating cleavage of C5 and increases in C5a levels, leading to an amplification loop of ANCA-mediated neutrophil activation42,43. In animal models, both C5a knockout and blockade of the neutrophil C5a receptor have been shown to be protective against ANCA-induced glomerulonephritis44.\n\nAn orally administered inhibitor of C5a receptor (CCX168) has recently completed phase II investigation in Europe (CLEAR: ClinicalTrials.gov identifier NCT01363388). The CLEAR study evaluated three arms (Table 1). All patients received standard induction with either CYC or RTX and were randomized to one of the following: CCX168 + low-dose (20 mg) initial prednisone, CCX168 + no initial prednisone, or placebo + high-dose (60 mg) initial prednisone. Preliminary data show that both treatment groups receiving CCX168 were non-inferior to the standard induction and high-dose prednisone. This was demonstrated by complete remission obtained at 12 weeks in 75% AAV patients in the high-dose prednisone + placebo group, 86% in the CCX168 + low-dose prednisone (p=0.005 for non-inferiority), and 81% in the CCX168 + no prednisone group (p=0.02 for non-inferiority). While this is clearly an important milestone for complement-targeted therapies, complete data analysis is yet forthcoming and the North American phase II trial of low- vs. high-dose CCX168 in addition to standard induction in AAV is still ongoing (CLASSIC: ClinicalTrials.gov identifier NCT02222155).\n\nFinally, evidence is accumulating that inflammatory cytokines may play a role in the pathogenesis and activity of AAV. Interleukin (IL)-6 levels are increased in both serum and histopathologic samples from patients with active AAV45. Furthermore, higher serum IL-6 levels appear to be associated with patients with frequent relapse and more severe organ damage45. IL-6 blockade with tocilizumab has shown promise in limited case reports45,46 and requires further evaluation. Similar to IL-6, increased serum levels of IL-17 and IL-23 have been observed in patients with AAV compared to healthy controls47. Additionally, AAV patients with higher IL-23 levels had more active disease and higher ANCA titers47. Both cytokines are associated with T helper 17 (Th17) cells, a T cell subset critical in mediating autoimmune disease. Specifically, IL-23 enhances the differentiation of T cells towards the Th17 subset and further assists in maintaining the production of IL-17, which itself is a proinflammatory cytokine with pleiotropic action. Ustekinumab (anti-IL-12/23) and secukinumab (anti-IL-17A) are both currently commercially available for the treatment of plaque psoriasis. These targeted anti-inflammatory cytokine therapies have yet to be trialed in AAV, but plans for future research are being considered.\n\n\nConclusions\n\nThe success of RTX in AAV is unequivocal, and its rapid utilization in these conditions highlights the four-decade-long unmet need for reliable alternatives to CYC. Induction with RTX can be considered a first-line option for both treatment-naïve and relapsing patients, particularly in young patients wanting to preserve fertility and for patients with a history, or at increased risk, of malignancy. The benefit of PLEX in induction management is still unclear but is considered reasonable in patients presenting with severe renal dysfunction. Although prospective trial evidence is limited, it is clear that maintenance treatment is needed following RTX induction in the majority of patients. In the next 5 years, the results from ongoing clinical trials will provide guidance and clarity to several clinical questions that have arisen in the post-RTX era.\n\nParalleling the advances made in rheumatoid arthritis treatment over the past two decades, the treatment of vasculitis in general, and AAV in particular, has now entered the increasingly expansive arena of targeted therapeutics. While phenotypically similar, the immune pathway targets at the individual level in immune-mediated diseases are extraordinarily complex and likely the reason for the variability in clinical outcome among large therapeutic trials. Significant advances have been made in understanding AAV at a population level, and this will ultimately lead to better understanding at the individual level to allow tailoring of treatments and a more personalized approach to management. In addition to generating novel therapeutics to newly identified pathway targets, the next decade will also likely bring combined therapeutic approaches where biologic agents are trialed in combination or succession.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nJayne D, Rasmussen N, Andrassy K, et al.: A randomized trial of maintenance therapy for vasculitis associated with antineutrophil cytoplasmic autoantibodies. N Engl J Med. 2003; 349(1): 36–44. PubMed Abstract | Publisher Full Text\n\nde Groot K, Harper L, Jayne DR, et al.: Pulse versus daily oral cyclophosphamide for induction of remission in antineutrophil cytoplasmic antibody-associated vasculitis: a randomized trial. Ann Intern Med. 2009; 150(10): 670–80. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHarper L, Morgan MD, Walsh M, et al.: Pulse versus daily oral cyclophosphamide for induction of remission in ANCA-associated vasculitis: long-term follow-up. Ann Rheum Dis. 2012; 71(6): 955–60. 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PubMed Abstract | Publisher Full Text\n\nBesada E, Koldingsnes W, Nossent JC: Long-term efficacy and safety of pre-emptive maintenance therapy with rituximab in granulomatosis with polyangiitis: results from a single centre. Rheumatology (Oxford). 2013; 52(11): 2041–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRhee EP, Laliberte KA, Niles JL: Rituximab as maintenance therapy for anti-neutrophil cytoplasmic antibody-associated vasculitis. Clin J Am Soc Nephrol. 2010; 5(8): 1394–400. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuillevin L, Pagnoux C, Karras A, et al.: Rituximab versus azathioprine for maintenance in ANCA-associated vasculitis. N Engl J Med. 2014; 371(19): 1771–80. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPendergraft WF 3rd, Cortazar FB, Wenger J, et al.: Long-term maintenance therapy using rituximab-induced continuous B-cell depletion in patients with ANCA vasculitis. Clin J Am Soc Nephrol. 2014; 9(4): 736–44. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRoubaud-Baudron C, Pagnoux C, Méaux-Ruault N, et al.: Rituximab maintenance therapy for granulomatosis with polyangiitis and microscopic polyangiitis. J Rheumatol. 2012; 39(1): 125–30. PubMed Abstract | Publisher Full Text\n\nPusey CD, Rees AJ, Evans DJ, et al.: Plasma exchange in focal necrotizing glomerulonephritis without anti-GBM antibodies. Kidney Int. 1991; 40(4): 757–63. PubMed Abstract | Publisher Full Text\n\nJayne DR, Gaskin G, Rasmussen N, et al.: Randomized trial of plasma exchange or high-dosage methylprednisolone as adjunctive therapy for severe renal vasculitis. J Am Soc Nephrol. 2007; 18(7): 2180–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWalsh M, Casian A, Flossmann O, et al.: Long-term follow-up of patients with severe ANCA-associated vasculitis comparing plasma exchange to intravenous methylprednisolone treatment is unclear. Kidney Int. 2013; 84(2): 397–402. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWalsh M, Catapano F, Szpirt W, et al.: Plasma exchange for renal vasculitis and idiopathic rapidly progressive glomerulonephritis: a meta-analysis. Am J Kidney Dis. 2011; 57(4): 566–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKlemmer PJ, Chalermskulrat W, Reif MS, et al.: Plasmapheresis therapy for diffuse alveolar hemorrhage in patients with small-vessel vasculitis. Am J Kidney Dis. 2003; 42(6): 1149–53. PubMed Abstract | Publisher Full Text\n\nCartin-Ceba R, Diaz-Caballero L, Al-Qadi MO, et al.: Diffuse Alveolar Hemorrhage Secondary to Antineutrophil Cytoplasmic Antibody-Associated Vasculitis: Predictors of Respiratory Failure and Clinical Outcomes. Arthritis Rheumatol. 2016; 68(6): 1467–76. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nOta M, Duong BH, Torkamani A, et al.: Regulation of the B cell receptor repertoire and self-reactivity by BAFF. J Immunol. 2010; 185(7): 4128–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSalazar-Camarena DC, Ortiz-Lazareno PC, Cruz A, et al.: Association of BAFF, APRIL serum levels, BAFF-R, TACI and BCMA expression on peripheral B-cell subsets with clinical manifestations in systemic lupus erythematosus. Lupus. 2016; 25(6): 582–92. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKrumbholz M, Specks U, Wick M, et al.: BAFF is elevated in serum of patients with Wegener's granulomatosis. J Autoimmun. 2005; 25(4): 298–302. PubMed Abstract | Publisher Full Text\n\nHolden NJ, Williams JM, Morgan MD, et al.: ANCA-stimulated neutrophils release BLyS and promote B cell survival: a clinically relevant cellular process. Ann Rheum Dis. 2011; 70(12): 2229–33. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWilde B, Thewissen M, Damoiseaux J, et al.: T cells in ANCA-associated vasculitis: what can we learn from lesional versus circulating T cells? Arthritis Res Ther. 2010; 12(1): 204. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLangford CA, Monach PA, Specks U, et al.: An open-label trial of abatacept (CTLA4-IG) in non-severe relapsing granulomatosis with polyangiitis (Wegener's). Ann Rheum Dis. 2014; 73(7): 1376–9. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nPerenyei M, Jayne DR, Floßmann O: Gusperimus: immunological mechanism and clinical applications. Rheumatology (Oxford). 2014; 53(10): 1732–41. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBirck R, Warnatz K, Lorenz HM, et al.: 15-Deoxyspergualin in patients with refractory ANCA-associated systemic vasculitis: a six-month open-label trial to evaluate safety and efficacy. J Am Soc Nephrol. 2003; 14(2): 440–7. PubMed Abstract | Publisher Full Text\n\nFlossmann O, Baslund B, Bruchfeld A, et al.: Deoxyspergualin in relapsing and refractory Wegener's granulomatosis. Ann Rheum Dis. 2009; 68(7): 1125–30. PubMed Abstract | Publisher Full Text\n\nSchmitt WH, Birck R, Heinzel PA, et al.: Prolonged treatment of refractory Wegener's granulomatosis with 15-deoxyspergualin: an open study in seven patients. Nephrol Dial Transplant. 2005; 20(6): 1083–92. PubMed Abstract | Publisher Full Text\n\nFlossmann O, Jayne DR: Long-term treatment of relapsing Wegener's granulomatosis with 15-deoxyspergualin. Rheumatology (Oxford). 2010; 49(3): 556–62. PubMed Abstract | Publisher Full Text\n\nSchreiber A, Xiao H, Jennette JC, et al.: C5a receptor mediates neutrophil activation and ANCA-induced glomerulonephritis. J Am Soc Nephrol. 2009; 20(2): 289–98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHao J, Meng LQ, Xu PC, et al.: p38MAPK, ERK and PI3K signaling pathways are involved in C5a-primed neutrophils for ANCA-mediated activation. PLoS One. 2012; 7(5): e38317. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXiao H, Dairaghi DJ, Powers JP, et al.: C5a receptor (CD88) blockade protects against MPO-ANCA GN. J Am Soc Nephrol. 2014; 25(2): 225–31. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBerti A, Cavalli G, Campochiaro C, et al.: Interleukin-6 in ANCA-associated vasculitis: Rationale for successful treatment with tocilizumab. Semin Arthritis Rheum. 2015; 45(1): 48–54. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTakenaka K, Ohba T, Suhara K, et al.: Successful treatment of refractory aortitis in antineutrophil cytoplasmic antibody-associated vasculitis using tocilizumab. Clin Rheumatol. 2014; 33(2): 287–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nNogueira E, Hamour S, Sawant D, et al.: Serum IL-17 and IL-23 levels and autoantigen-specific Th17 cells are elevated in patients with ANCA-associated vasculitis. Nephrol Dial Transplant. 2010; 25(7): 2209–17. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "14464",
"date": "20 Jun 2016",
"name": "Cees GM Kallenberg",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14465",
"date": "20 Jun 2016",
"name": "Charles Pusey",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1436
|
https://f1000research.com/articles/5-1412/v1
|
17 Jun 16
|
{
"type": "Review",
"title": "Recent advances in primary Sjogren's syndrome",
"authors": [
"Nicholas Holdgate",
"E. Wiliam St.Clair",
"Nicholas Holdgate"
],
"abstract": "Primary Sjögren’s syndrome, a chronic inflammatory process, is among the most commonly occurring rheumatologic diseases. The clinical hallmark of this disease is exocrine gland dysfunction, resulting predominately in dry eyes and dry mouth. However, the disease often extends beyond the exocrine glands to seriously affect other organs systems, such as the lungs, kidneys, and nervous system. Moreover, patients with primary Sjögren’s syndrome develop non-Hodgkin’s B cell lymphoma at a substantially higher rate than the general population. New research has improved our understanding of disease mechanisms, with notable advances in our knowledge about the genetic susceptibility of disease, the molecular details of the chronic inflammatory response in the salivary glands, and the complex role of the type 1 interferon pathway. The pipeline of drugs under development for the treatment of primary Sjögren’s syndrome is enriched with novel biologics and small molecular entities targeting the pathogenic process. Herein, we summarize the latest advances in elucidating the pathogenesis of primary Sjögren’s syndrome and highlight new drugs in clinical development aiming to reverse the glandular dysfunction and favorably impact the systemic features of this disease.",
"keywords": [
"Sjögren’s syndrome",
"inflammatory",
"rheumatologic diseases"
],
"content": "Introduction\n\nDespite its prevalence among the rheumatologic diseases, primary Sjögren’s syndrome has attracted less attention from researchers compared to rheumatoid arthritis (RA) and systemic lupus erythematous (SLE), partly owing to a previous misconception that the disease was mostly a nuisance without recognizing that it can threaten the function of vital organ systems. The clinical hallmarks of Sjögren’s syndrome have been well known for many decades and include keratoconjunctivitis sicca, or dry eyes, and xerostomia, or dry mouth. Sjögren’s syndrome is termed secondary when the sicca symptoms occur in association with RA, SLE, or other chronic inflammatory disease. Otherwise, it is termed primary Sjögren’s syndrome. The clinical diagnosis of primary Sjögren’s syndrome is based on the presence of symptoms of dry eyes and dry mouth, often with objective evidence of keratoconjunctivitis sicca and/or decreased salivary flow, and a positive test for serum anti-Ro antibodies or rheumatoid factor or, in the absence of these autoantibodies, a labial salivary gland biopsy showing focal lymphocytic infiltrates1. Reversal of glandular dysfunction and management of extraglandular disease has been hampered by the lack of a proven disease-modifying therapy. Herein, we describe some of the exciting new research about the genetic and immunologic mechanisms of primary Sjögren’s syndrome and the recent progress towards finding a disease-modifying therapy.\n\nPrimary Sjögren’s syndrome is among the most common of the systemic autoimmune diseases, ranging in prevalence from 0.1 to 0.6%; it is much more common in women than in men2. The disease is termed an “exocrinopathy” because of its predominant effects on the lacrimal and salivary glands, as well as other exocrine glands in the larynx (hoarseness), trachea (cough), skin (pruritus), and vagina (dyspareunia). In addition to the exocrinopathy, up to 75% of patients with primary Sjögren’s syndrome suffer from extraglandular disease manifestations, and up to 25% develop moderate or severe extraglandular disease3. Extraglandular manifestations range from the less serious complications of purpura, urticaria, inflammatory arthritis, and Raynaud’s syndrome to the more severe end of the spectrum exemplified by renal disease, lung disease, and peripheral neuropathy. Notably, patients with primary Sjögren’s syndrome have an increased risk of developing non-Hodgkin B cell lymphoma, with a recent study showing a cumulative risk at 15 years after diagnosis of 9.8%; this risk is higher than that of patients with RA and SLE who also have an increased risk of developing non-Hodgkin lymphoma4,5. Interestingly, most non-Hodgkin B cell lymphomas that develop in patients with primary Sjögren’s syndrome belong to the marginal zone histological type and localize to the mucosa-associated lymphoid tissue (MALT), where the underlying disease is most active. The presence of parotid gland enlargement, rheumatoid factor, low C4, cryoglobulinemia, lymphopenia, and higher levels of disease activity as measured by the EULAR Sjögren’s syndrome disease activity index (ESSDAI) predicts a higher lymphoma risk6.\n\n\nCurrent management\n\nIn primary Sjögren’s syndrome, the current treatments aim to reduce symptoms of the exocrinopathy as well as control the extraglandular features of the disease. Symptomatic therapies include topical therapies, such as artificial tears, artificial saliva, nasal saline spray, vaginal estrogen cream, and moisturizing skin lotions. Secretagogues, such as sugarless candy and chewing gum, as well as the muscarinic receptor agonists cevimeline and pilocarpine may be used to stimulate tear and saliva production. Topical cyclosporine eye drops are efficacious for improving the signs and symptoms of dry eyes.\n\nAt this stage, no disease-modifying drugs have been shown in randomized, placebo-controlled trials to be effective for the treatment of primary Sjögren’s syndrome. While hydroxychloroquine has been employed for reducing the symptoms of fatigue and joint pain in primary Sjögren’s syndrome, it failed in a randomized controlled trial to significantly reduce symptoms of dryness, pain, and fatigue over placebo7. Methotrexate has not been well studied for the treatment of primary Sjögren’s syndrome. In a small open study published 20 years ago, oral weekly methotrexate was not effective in increasing tear or saliva production8. More recently, the tumor necrosis factor (TNF)-α inhibitors infliximab and etanercept have been investigated for their efficacy and safety in patients with primary Sjögren’s syndrome and neither was found to be effective for improving disease outcomes9,10.\n\nThe dearth of proven effective therapies for primary Sjögren’s syndrome has forced clinicians to take an empiric approach to the systemic treatment of extraglandular disease, guided by the severity of organ system involvement. Mild sensory neuropathies, common in patients with primary Sjögren’s syndrome, are generally treated symptomatically with, for example, gabapentin or pregabalin. Recurrent lower extremity purpura caused by a leukocytoclastic vasculitis may usually be managed with support stockings and non-steroidal anti-inflammatory drugs. Systemic corticosteroids may be prescribed for the treatment of organ-threatening vasculitis, non-specific interstitial pneumonitis (NSIP), interstitial nephritis, glomerulonephritis, and motor neuropathies. Other immunosuppressive agents, such as azathioprine, mycophenolate mofetil, and cyclophosphamide, are usually reserved for the treatment of serious or life-threatening extraglandular disease, such as NSIP and glomerulonephritis.\n\n\nAdvances in understanding of disease pathogenesis\n\nPrimary Sjögren’s syndrome results from a complex interplay of several factors, including genetic and epigenetic controls of immune homeostasis and gene expression, age and gender, and environmental insults. It follows a typical multistep model of human autoimmune diseases characterized by loss of immunologic tolerance to self-antigens, the permissive production of autoantibodies, and the subsequent emergence of disease11,12. Studies of labial salivary glands reveal the contribution of a panoply of cell types, including T cells, B cells, macrophages, dendritic cells, and epithelial cells, that combine to orchestrate a persistent chronic inflammatory response.\n\nThe basis for the exocrine gland dysfunction is poorly understood and may result from effects related to the pro-inflammatory cytokine/chemokine milieu, autoantibody-mediated blockade of muscarinic acetylcholine receptors (e.g. anti-muscarinic receptor 3 antibodies), neurotransmitter imbalances, or the destruction of glands. The potential reversibility of glandular dysfunction is unclear and must necessarily await the discovery of a disease-modifying drug that can ameliorate the loss of lacrimal and salivary secretory flow.\n\nNewly discovered genetic associations in primary Sjögren’s syndrome implicate disturbances in both the adaptive and the innate immune pathways13,14. Genome-wide association and candidate gene studies have identified susceptibility loci in BLK and CXCR5, genes encoding proteins involved in B cell activation, trafficking, and spatial localization, and in STAT4, IL-12A, and IRF5, genes regulating both adaptive and innate immunity. The finding of risk variants in the IRF5 gene may explain in part the activation of the type 1 interferon (IFN) pathway in primary Sjögren’s syndrome15, as IRF5 encodes a transcription factor important for the production of type 1 IFN16. A risk variant of IRF5, a CGGGG indel, has been associated with increased levels of IRF5 transcripts, implying a functional role in the regulation of this pathway17. STAT4 is triggered by type 1 IFN, interleukin (IL)-12, and IL-23 and when activated leads to the induction of T helper type 1 (TH1) cells and up-regulation of IFN-γ.\n\nGenome-wide association and candidate gene studies have also identified risk loci in the TNFAIP3 and TNIP genes, which regulate nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling. The potential importance of the TNFAIP3 locus in the pathogenesis of primary Sjögren’s syndrome is underscored by the finding that TNFAIP3 knockout mice develop an autoimmune phenotype and have high rates of lymphoma18. Moreover, a study of patients with primary Sjögren’s syndrome from a French cohort has recently found germline mutations in the coding region of TNFAIP3 (A20) that are associated with an increased risk for lymphoma19. Interestingly, in some instances, the samples of lymphoma tissue harbored the same germline mutations in TNFAIP3 that were associated with an increased risk for lymphoma development, while in other cases without the germline mutation, the lymphoma tissue had somatic mutations in TNFAIP3. Some of these TNFAIP3 variants impaired control of NFκB activation, suggesting a possible escape mechanism for transition into lymphoma19.\n\nThe epigenetic regulation of gene expression in primary Sjögren’s syndrome has not been studied until recently. Patients with primary Sjögren’s syndrome have an increase in hypomethylation of the STAT4, IL-12A, IRF5, BLK, CXCR5, and TNIP genes20,21, suggesting a possible role for epigenetic regulation in the pathogenesis of this disease.\n\nThe role of B cells in the pathogenesis of primary Sjögren’s syndrome is strongly implied by the association of this disease with autoantibody production, B cell hyperactivity, germinal center formation in the target tissue, and lymphomagenesis. In primary Sjögren’s syndrome, it is not known if pathologic B cell activation occurs in the spleen and lymph nodes or the germinal center-like structures (tertiary lymphoid tissue) of the target tissues, or both. T-cell-dependent antigens that activate B cells rely on T follicular helper (TFH) cells, which stimulate the formation and maintenance of germinal centers through the expression of CD154 (CD40 ligand) on the cell surface and the secretion of IL-4 and IL-21; they mediate the selection and survival of B cells that differentiate into antibody-secreting plasma cells and memory B cells. TFH cells have been identified in labial salivary gland tissue, predominantly within organized structures22. The differentiation of CD4+ naïve T cells into TFH cells may be promoted by the glandular epithelium. Salivary gland epithelial cells expressing IL-6 and inducible T cell co-stimulator ligand (ICOS-L) have been shown in culture to stimulate the differentiation of CD4+ naïve T cells into IL-21-secreting TFH cells23.\n\nControlling TFH cell-mediated B cell activation may be a useful therapeutic strategy in primary Sjögren’s syndrome. Two experimental therapies, abatacept and rituximab, have been shown to reduce the absolute number of circulating TFH cells24, but the significance of these findings is unclear, since neither of these agents has been proven effective in randomized, controlled trials involving patients with primary Sjögren’s syndrome. In the future, inhibiting the differentiation of TFH cells by neutralizing IL-21 or IL-6 or blocking the interaction between ICOS and ICOS-L, either alone or in combination, may also be therapeutic strategies worth pursuing.\n\nB-cell-activating factor (BAFF), a member of the TNF receptor family, is a potent activator of B cells. BAFF is the natural ligand of three receptors, BAFF-R (BR3), a transmembrane activator and calcium modulator, as well as transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA). Several lines of evidence implicate BAFF in the pathogenesis of primary Sjögren’s syndrome. First, compared with healthy controls, patients with primary Sjögren’s syndrome have higher serum levels of BAFF and show increased expression of BAFF in labial salivary glands25. Second, BAFF transgenic mice exhibit a Sjögren’s syndrome phenotype characterized by infiltration of lacrimal and salivary glands with lymphocytes that include a unique population of marginal zone B cells26. Third, salivary gland epithelial cells from patients with primary Sjögren’s syndrome also express BAFF; in addition, type 1 IFN, which is up-regulated in both blood and salivary gland tissue from patients with primary Sjögren’s syndrome, is a potent stimulator of BAFF expression25,27. BAFF engagement with its receptor, BR3, has been shown to activate the phosphatidylinositol 3-kinase delta isoform (PI3kδ), a signaling molecule involved in the regulation of cell growth, proliferation, differentiation, motility, survival, and intracellular trafficking of B cells, as well as lymphomagenesis28. For these reasons, the BAFF/BR3 pathway is a promising therapeutic target in primary Sjögren’s syndrome.\n\nThe lymphotoxin β receptor (LTβR) is part of the signaling system that directs stromal cells to differentiate into specialized vasculature and regulates certain reticular networks that guide and position cells for optimal encounters with antigen29. Blockade of the LTβR system in mice reduces addressin expression on high endothelial venules (HEVs) and thereby inhibits entry of lymphocytes into lymph nodes and mucosal environments, resulting in a circulating lymphocytosis30. In male NOD mice, LTβR-Ig treatment reduces glandular inflammation, blocks HEV formation, and partially restores salivary flow31. In a phase II trial, baminercept (LTβR-Ig) treatment was ineffective in decreasing the signs and symptoms of RA; however, it was shown to increase the blood lymphocyte counts and reduce the whole blood IFN signature32. Although inhibiting the lymphotoxin pathway has a sound theoretical basis for treating primary Sjögren’s syndrome, preliminary results from a randomized, placebo-controlled 24-week trial showed no significant differences between the baminercept and placebo groups in the change in stimulated salivary flow or ESSDAI33.\n\nViruses have long been considered etiologic factors in primary Sjögren’s syndrome. They have been receiving more attention of late as a possible disease trigger because of the association between primary Sjögren’s syndrome and up-regulation of the type 1 IFN pathway, an innate immune mechanism of antiviral host defense. An IFN signature has been detected in the peripheral blood of 65% of patients with primary Sjögren’s syndrome34 and can be observed in labial salivary gland tissue from patients with this disease34. The majority of the IFN inducible genes are up-regulated by both IFN-α and IFN-γ, while some are uniquely due to the effects of IFN-α or IFN-γ. In the labial salivary glands, there is heterogeneity among patients in the expression of IFN-α and IFN-γ inducible genes, with higher focus scores associated with predominant IFN-γ effects34,35.\n\nTo protect against viral infection, the host innate immune system has evolved sensors of nucleic acids. The same nucleic acid sensors that defend against viruses may also contribute to the pathogenesis of autoimmune diseases. The two main systems for detecting nucleic acids are the Toll-like receptors (TLRs) and the cytosolic RNA and DNA sensors. The endosomal TLRs mainly detect nucleic acids, such as double-stranded RNA (TLR3), single-stranded RNA (TLR7), and double-stranded DNA (TLR9)36. Through an autoantibody-dependent, FcγR-mediated process, nucleic acids gain access to the cytosol and in turn ligate endosomal TLRs, which activate dendritic cells and macrophages to produce type 1 IFN37. Two other key intracellular sensors of RNA and DNA may also play a role in the pathogenesis of autoimmune diseases such as primary Sjögren’s syndrome, namely the retinoic acid-inducible gene I (RIG-I)-like receptors RIG-I and Mda5 and cyclic GMP-AMP synthase/STING, respectively, which also activate the production of type 1 IFN38. It has been theorized that the evolutionary pressures to produce a more robust antiviral response may also tip the balance towards developing an autoimmune disease.\n\nEpstein-Barr virus (EBV) is a promising candidate for triggering autoimmune disease owing to its ability to infect B cells and promote the survival of autoreactive B cell clones39. A human DNA γ-herpesvirus, it establishes an asymptomatic latent infection in B cells and epithelial cells. EBV-encoded small RNA (EBER), the most abundant transcripts in latently infected cells, contribute to EBV-mediated pathogenesis by activating RIG-I and stimulating innate immune signaling through TLR340. EBERs also bind to the La protein and are secreted outside the cell in exosomes, where they may bind to TLR3 and activate dendritic cells41. However, studies comparing the expression of EBV nucleic acids and proteins in the salivary glands between patients with primary Sjögren’s syndrome and healthy controls have produced conflicting results.\n\nCroia and colleagues recently attempted to settle this controversy by focusing their search for latent EBV infection on labial salivary gland tissue with germinal center-like structures expressing the mRNA for activation-induced cytidine deaminase (AID) and CD2142. AID is involved in somatic hypermutation, gene conversion, and class-switch recombination of immunoglobulin genes, while CD21 is a marker of follicular dendritic cells. They found that EBERs were preferentially expressed in the AID+CD21+ labial salivary gland biopsies compared to AID-CD21- samples from the disease patients and healthy controls, with EBERs localized mostly to the follicular B cells and plasma cells. Further evidence of EBV latency was obtained by detection of the latent EBV antigen LMP-2 in the B cells within the ectopic follicles. Notably, the lytic-phase antigen BFRF1 was expressed in the perifollicular plasma cells with Ro52 autoreactivity, a prominent autoantigen in primary Sjögren’s syndrome. Whether EBER transcripts from latent EBV infection are contributing to the inflammatory response in primary Sjögren’s syndrome is an open question, but the results of this study draw more attention to this possibility.\n\nRetrotransposons comprise approximately 40% of the human genome and include the retrovirus long-terminal repeats (LTRs), members of the ‘long interspersed nuclear element 1’ (LINE-1, or L1) family, and ‘short interspersed nuclear elements’ (SINEs)39. Most human SINEs are ‘Alu’ elements, which are capable of replicating themselves by hijacking the L1 reverse transcriptase39. A recent study found that anti-Ro60–positive SLE immune complexes containing Alu RNAs and Alu transcripts were up-regulated in whole blood samples from patients with SLE relative to control samples and established that aberrant expression of endogenous Alu RNAs stimulated a TLR7-dependent response that enhances the secretion of IFN-α, IL-6, and TNF-α41. The results from these studies suggest that retroelements may stimulate autoimmunity by amplifying immune responses through endogenous RNA sensors41. There is also evidence that endogenous retroviruses and innate sensing pathways are integral to T cell-independent B cell activation43. Thus, if retroelements play a significant role in the pathogenesis of primary Sjögren’s syndrome, then decreasing the abundance of nucleic acids inside cells or increasing the threshold of their intracellular sensors may represent a new strategy for treating this disease.\n\nNatural killer (NK) cells are innate lymphoid cells that play an important role in antiviral host defense and are increasingly implicated in the pathogenesis of autoimmune disease44. Human NK cells are CD3- and separated into two major groups by their expression of CD16 and CD56. The CD56dim NK cells that comprise approximately 90% of the circulating NK cells express high levels of CD16, inhibitory killer-like receptors (KIRs), and perforin; CD56bright NK cells express low levels of CD16, KIRs, and perforin and are abundant in secondary lymphoid tissues where they modulate immune responses through their secretion of cytokines and chemokines44.\n\nNKp30, a NK cell-specific activating receptor, regulates cross-talk between NK and dendritic cells and mediates secretion of IL-12 and IFN-γ. In a large cohort of patients with primary Sjögren’s syndrome, genetic polymorphisms within the promoter region of the NKp30 gene were found to be associated with decreased disease susceptibility and reduced levels of gene expression and function of the NKp30 protein45. In this study, circulating NK cells from the patients with primary Sjögren’s syndrome expressed higher levels of NKp30 than controls and showed increased secretion of IFN-γ45. Moreover, excessive numbers of NK cells were found to be present in the labial salivary gland tissue from patients with primary Sjögren’s syndrome, localizing outside inflammatory foci, and nearby glandular epithelial cells expressing B7H6, the ligand for NKp3045. It is possible that NKp30+ cells may be an important source of IFN-γ in the target organ of primary Sjögren’s syndrome through the engagement of B7H6 on epithelial cells.\n\nIL-22, a member of the IL-1 family, is produced by a variety of cell types, including TH17 cells, γδ T cells, NK T cells, and innate lymphoid cells (ILCs); it mainly acts on nonhematopoietic cells such as epithelial cells46. Increased expression of IL-22 has been linked to the pathogenesis of tissue inflammation and regeneration of epithelial tissues following injury46. Therefore, IL-22 exerts both pro-inflammatory and protective effects and its function has been shown to be tissue and context dependent. IL-22, IL-23, and IL-17 mRNA and protein levels are up-regulated in labial salivary gland tissue from patients with primary Sjögren’s syndrome compared to healthy controls47.\n\nIn labial salivary glands from patients with primary Sjögren’s syndrome, IL-22 has been shown to be produced by activated dendritic cells, T cells, and the NKp44+ subset of NK cells47. Further insights into the possible role of IL-22 have been investigated in a virus-induced mouse model of Sjögren’s syndrome, where IL-22 was found to be mainly produced in the early stages of infection by γδ T cells and then later by αβ T cells, with lesser amounts coming from ILCs and NK cells48. In this study, IL-22/IL-22R engagement was needed for enhanced expression of CXCL12 and CXCL13, recruitment of B cells, and organization of tertiary lymphoid tissue. Although IL-22 may be a potential therapeutic target in primary Sjögren’s syndrome, its role in promoting tissue regeneration and restoring barrier function after tissue injury may preclude systemic IL-22 blockade over long periods of time.\n\n\nInvestigational systemic therapies\n\nThe pipeline of investigational therapies for primary Sjögren’s syndrome has grown substantially over the past 5 years (Table 1). Despite the initial promise of rituximab therapy for this disease, the results of two randomized, placebo-controlled trials from France and the United Kingdom recently failed to demonstrate significant improvement in the endpoints of dryness, pain, and fatigue49,50. Other targeted approaches are in much earlier phases of development and have not been adequately studied in randomized, controlled trials. Open treatment with belimumab, a monoclonal antibody that inhibits BAFF, led to some improvement in physician assessment of systemic disease activity and serologic markers of B cell function but failed to show any significant benefit in terms of reducing symptoms of fatigue, pain, and dryness51.\n\n* From clinicaltrials.gov\n\nAbbreviations: BAFF, B-cell-activating factor; BAFF-R, B-cell-activating factor receptor; CTLA4-Ig, cytotoxic T-lymphocyte-associated protein 4 – Ig fusion protein (abatacept); ICOS, inducible T cell costimulator; IL-6, interleukin-6; IL-6R, interleukin-6 receptor; PI3Kδ, phosphatidylinositol-4,5-bisphosphate 3-kinase delta; TFH, T follicular helper cell; TH17, T helper 17 cell; Treg cell, T regulatory cell\n\nA clinical trial has been initiated in patients with primary Sjögren’s syndrome that combines treatment with both rituximab and belimumab. It is based on the rationale that BAFF levels increase following rituximab-induced CD20 B cell depletion52, which in turn may promote the survival of self-reactive B cells and perpetuate the breach in B cell tolerance. In theory, combining belimumab with a B-cell-depleting agent would neutralize the excessive BAFF and allow for reconstitution of a non-self-reactive B cell repertoire. Other inhibitors of the BAFF/BR3 pathway are under development as well. Since PI3kδ is a signaling molecule crucial for BR3 function28, studies are also now underway in patients with primary Sjögren’s syndrome to investigate the potential benefits of a small molecule PI3kδ inhibitor.\n\nBruton’s tyrosine kinase (Btk) inhibitors, which are approved for the treatment of certain B cell malignancies, are also in early stage clinical trials for the treatment of primary Sjögren’s syndrome. These small molecule Btk inhibitors can not only inhibit B cell receptor-dependent B cell proliferation and activation but also impact FcR-dependent proinflammatory cytokine production by myeloid cells53.\n\nThe use of co-stimulatory blockade with abatacept is a well-reasoned therapeutic intervention for primary Sjögren’s syndrome owing to the important role of T cell activation in the pathogenesis of this disease. In an open-label study, treatment with abatacept was shown to significantly decrease the scores for the ESSDAI and EULAR Sjögren’s syndrome patient-reported index (ESSPRI) and the serum levels of rheumatoid factor and IgG54. However, measures of salivary and lacrimal gland function did not change during treatment52. Larger controlled studies of abatacept therapy for primary Sjögren’s syndrome are in progress.\n\nSeveral biologics are under development for the treatment of primary Sjögren’s syndrome that inhibit the activation of T cells. A non-depleting, Fc-silent, anti-CD40 monoclonal antibody (CFZ533) blocks the binding of CD40 to CD154 and thereby inhibits T cell activation. CD154 is particularly important in the activation of TFH cells. TFH cells are also being targeted by a monoclonal antibody to B7RP1 (ICOS-L), or AMG557. Blocking the B7RP1/ICOS-L pathway has been shown to decrease the differentiation of CD4+ naïve T cells into TFH cells and inhibit germinal center formation55 and thus could decrease B cell activation and ectopic lymphoid tissue formation in the target organs of primary Sjögren’s syndrome.\n\nTocilizumab, an IL-6 receptor-blocking antibody, has been approved for the treatment of RA and juvenile idiopathic arthritis and is currently in a phase II study investigating its efficacy and safety in primary Sjögren’s syndrome. The IL-6/IL-6 receptor pathway is important in promoting the differentiation and maintenance of TH17 and TFH cells. Low-dose IL-2 therapy is also being considered as a possible therapy for primary Sjögren’s syndrome because of its capacity to expand the T regulatory cell population56,57; however, IL-2 can also have the undesirable effect of activating T cells and perpetuating the effector arm of a T-cell-driven response. Whether the dose of IL-2 can be adjusted to favor the expansion of T regulatory cells over the activation of pathogenic effector T cells will require further study.\n\nIFN-α is an intriguing therapeutic target in primary Sjögren’s syndrome for the reasons described above. Sifalimumab and rontalizumab, anti-IFN-α monoclonal antibodies under development for the treatment for SLE, would also have a strong rationale for testing in primary Sjögren’s syndrome. In addition, anti-retroviral therapies might have some therapeutic appeal in the future considering the emerging evidence that expression of endogenous retroelements in the target tissue may engage nucleic acid sensors and augment chronic inflammatory responses.\n\n\nLooking forward\n\nDrug developers are gradually shifting their attention towards primary Sjögren’s syndrome as a possible indication for new targeted therapies. The prevalence of primary Sjögren’s syndrome and the ready access to the target tissue through labial salivary gland biopsy provides unique advantages for investigation. Moreover, the lack of an approved disease-modifying therapy affords appropriate equipoise for conducting placebo-controlled trials of experimental therapies without the concomitant use of background immunosuppressive agents.\n\nThere are also challenges ahead in bringing a new drug through the development process owing to the lack of a roadmap for success. Many failed drug trials in primary Sjögren’s syndrome have raised questions about the optimal approach for investigating new therapeutics in the future. Thus far, the focus has been on interventions that are immunomodulatory and that down-regulate chronic inflammatory responses. Other mechanisms may be at play, such as neuroendocrine abnormalities, that affect some of the disease manifestations, such as fatigue. There is uncertainty about the most appropriate selection of subjects for clinical trials. Some authorities have advocated for the inclusion of patients with early disease only (e.g. < 4 years from diagnosis) with a minimum cut-off for stimulated whole salivary flow to ensure participants have sufficient glandular secretory capacity to detect a treatment response. This approach is logical if the experimental treatment is aimed at improving glandular function. Other trials may be designed to focus on the subset of patients with moderate-to-high levels of systemic disease activity if the investigational drug is hypothesized to benefit patients with extraglandular manifestations of disease.\n\nWhat is the appropriate primary endpoint for evaluating the clinical efficacy of a targeted therapy? Is it unstimulated or stimulated salivary flow? The answer is probably “yes” if increasing tear and salivary flow is the postulated action of the experimental agent. If the therapy is aimed at other disease manifestations, then two composite indices, the ESSPRI and ESSDAI, have been developed that broadly quantify disease activity. ESSPRI is an instrument that measures patient-oriented outcomes such as dryness, fatigue and pain symptoms, while the ESSDAI is a validated index of systemic disease activity. Recent work has set thresholds for clinically meaningful improvement in the ESSPRI (decrease in one point or 15%) and ESSDAI (≥ three points)58. It has been proposed that an ESSDAI of five points or more (moderate disease activity) should be an eligibility requirement for evaluating new therapies directed at systemic manifestations of primary Sjögren’s syndrome58. However, applying these restricted inclusion criteria significantly reduces the proportion of patients with primary Sjögren’s syndrome that would be eligible for a clinical trial59,60. Regardless, a more focused approach of testing new drugs in selected study populations with outcomes based on the mechanistic rationale of the experimental drug may improve the success rate of clinical trials in this disease.\n\nThe accelerated pace of discovery in primary Sjögren’s syndrome promises to illuminate the genetic, epigenetic, and environmental factors that are responsible for triggering and perpetuating the disease process. This new knowledge will inform the development of novel therapies that in the future will positively impact the care of patients who suffer from this chronic illness.\n\n\nAbbreviations\n\nAID, activation-induced cytidine deaminase; BAFF, B-cell-activating factor; BAFF-R, B-cell-activating factor receptor; BCMA, B cell maturation antigen; Btk, Bruton’s tyrosine kinase; EBV, Epstein-Barr virus; EBER, EBV encoded small RNA; ESSDAI, EULAR Sjögren’s syndrome disease activity index; ESSPRI, EULAR Sjögren’s syndrome patient-reported index; HEVs, high endothelial venules; ICOS, inducible T cell co-stimulator; ICOS-L, ICOS ligand; IFN, interferon; IL, interleukin; ILC, innate lymphoid cell; KIR, killer-like receptor; LINE-1/L1, long interspersed nuclear element 1; LTβR, lymphotoxin β receptor; LTRs, long-terminal repeats; MALT, mucosa-associated lymphoid tissue; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; NK, natural killer; NSIP, non-specific interstitial pneumonitis; PI3kδ, phosphatidylinositol 3-kinase delta; RA, rheumatoid arthritis; RIG-1, retinoic acid-inducible gene 1; SINEs, short interspersed nuclear elements; SLE, systemic lupus erythematous; TACI, transmembrane activator and calcium-modulator and cyclophilin ligand interactor; TFH, T follicular helper; TH, T helper; TNF, tumor necrosis factor; TLR, Toll-like receptor.",
"appendix": "Competing interests\n\n\n\nNicholas Holdgate declares no competing interests. Biogen provided study drug for a clinical trial in primary Sjogren's syndrome and in-kind support for mechanistic studies. E. William St. Clair also serves as a consultant for Bristol Myers Squibb.\n\n\nGrant information\n\nE. William St. Clair has received funding by NIAID grant 5U19-AI056363 (Autoimmunity Centers of Excellence) to support a clinical trial of baminercept in primary Sjögren’s syndrome.\n\n\nReferences\n\nRasmussen A, Ice JA, Li H, et al.: Comparison of the American-European Consensus Group Sjogren's syndrome classification criteria to newly proposed American College of Rheumatology criteria in a large, carefully characterised sicca cohort. Ann Rheum Dis. 2014; 73(1): 31–8. 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PubMed Abstract | Publisher Full Text\n\nLee EG, Boone DL, Chai S, et al.: Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice. Science. 2000; 289(5488): 2350–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNocturne G, Tarn J, Boudaoud S, et al.: Germline variation of TNFAIP3 in primary Sjögren's syndrome-associated lymphoma. Ann Rheum Dis. 2016; 75(4): 780–3. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nImgenberg-Kreuz J, Sandling JK, Carlsson Almlof J, et al.: Hypomethylation in Enhancer and Promoter Regions of Interferon Regulated Genes in Multiple Tissues Is Associated with Primary Sjögren’s Syndrome. [Abstract 2100]. Arthritis Rheumatol. 2015; 67(suppl 10). Reference Source\n\nCole M, Quach D, Quach HL, et al.: Genome-Wide DNA Methylation Signatures of Salivary Gland Inflammation in Sjogren’s Syndrome. [Abstract 1257]. Arthritis Rheumatol. 2015; 67(suppl 10). Reference Source\n\nSzabo K, Papp G, Dezso B, et al.: The histopathology of labial salivary glands in primary Sjögren's syndrome: focusing on follicular helper T cells in the inflammatory infiltrates. Mediators Inflamm. 2014; 2014: 631787. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nGong YZ, Nititham J, Taylor K, et al.: Differentiation of follicular helper T cells by salivary gland epithelial cells in primary Sjögren's syndrome. J Autoimmun. 2014; 51: 57–66. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nVerstappen GM, Meiners PM, Corneth OB, et al.: Treatment with Abatacept or Rituximab Targets T Follicular Helper Cells in Patients with Primary Sjogren s Syndrome. [Abstract 3205]. Arthritis Rheumatol. 2015; 67(suppl 10). Reference Source\n\nGroom J, Kalled SL, Cutler AH, et al.: Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren's syndrome. J Clin Invest. 2002; 109(1): 59–68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChiorini JA, Cihakova D, Ouellette CE, et al.: Sjögren syndrome: advances in the pathogenesis from animal models. J Autoimmun. 2009; 33(3–4): 190–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIttah M, Miceli-Richard C, Gottenberg JE, et al.: Viruses induce high expression of BAFF by salivary gland epithelial cells through TLR- and type-I IFN-dependent and -independent pathways. Eur J Immunol. 2008; 38(4): 1058–64. PubMed Abstract | Publisher Full Text\n\nNayar S, Campos J, Buckley C, et al.: Phosphatidylinositol-3-Kinase Delta Pathway a Novel Therapeutic Target for Sjogren’s Syndrome. [Abstract 1053]. Arthritis Rheumatol. 2015; 67(suppl 10). Reference Source\n\nBrowning JL: Inhibition of the lymphotoxin pathway as a therapy for autoimmune disease. Immunol Rev. 2008; 223(1): 202–20. PubMed Abstract | Publisher Full Text\n\nBrowning JL, Allaire N, Ngam-Ek A, et al.: Lymphotoxin-beta receptor signaling is required for the homeostatic control of HEV differentiation and function. Immunity. 2005; 23(5): 539–50. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGatumu MK, Skarstein K, Papandile A, et al.: Blockade of lymphotoxin-beta receptor signaling reduces aspects of Sjögren's syndrome in salivary glands of non-obese diabetic mice. Arthritis Res Ther. 2009; 11(1): R24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBienkowska J, Allaire N, Thai A, et al.: Lymphotoxin-LIGHT pathway regulates the interferon signature in rheumatoid arthritis. PLoS One. 2014; 9(11): e112545. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSt Clair EW, Baer AN, Noaiseh G, et al.: The Clinical Efficacy and Safety of Baminercept, a lymphotoxin-Beta Receptor Fusion Protein, in Primary Sjögren’s Syndrome: Results from a Randomized, Double-Blind, Placebo-Controlled Phase II Trial. [Abstract 3203]. Arthritis Rheumatol. 2015; 67(suppl 10). Reference Source\n\nHall JC, Casciola-Rosen L, Berger AE, et al.: Precise probes of type II interferon activity define the origin of interferon signatures in target tissues in rheumatic diseases. Proc Natl Acad Sci U S A. 2012; 109(43): 17609–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHall JC, Baer AN, Shah AA, et al.: Molecular Subsetting of Interferon Pathways in Sjögren's Syndrome. Arthritis Rheumatol. 2015; 67(9): 2437–46. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nClancy RM, Markham AJ, Buyon JP: Endosomal Toll-like receptors in clinically overt and silent autoimmunity. Immunol Rev. 2016; 269(1): 76–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLövgren T, Eloranta ML, Båve U, et al.: Induction of interferon-alpha production in plasmacytoid dendritic cells by immune complexes containing nucleic acid released by necrotic or late apoptotic cells and lupus IgG. Arthritis Rheum. 2004; 50(6): 1861–72. PubMed Abstract | Publisher Full Text\n\nVolkman HE, Stetson DB: The enemy within: endogenous retroelements and autoimmune disease. Nat Immunol. 2014; 15(5): 415–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPender MP: Infection of autoreactive B lymphocytes with EBV, causing chronic autoimmune diseases. Trends Immunol. 2003; 24(11): 584–8. PubMed Abstract | Publisher Full Text\n\nIwakiri D: Multifunctional non-coding Epstein-Barr virus encoded RNAs (EBERs) contribute to viral pathogenesis. Virus Res. 2016; 212: 30–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHung T, Pratt GA, Sundararaman B, et al.: The Ro60 autoantigen binds endogenous retroelements and regulates inflammatory gene expression. Science. 2015; 350(6259): 455–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCroia C, Astorri E, Murray-Brown W, et al.: Implication of Epstein-Barr virus infection in disease-specific autoreactive B cell activation in ectopic lymphoid structures of Sjögren's syndrome. Arthritis Rheumatol. 2014; 66(9): 2545–57. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nZeng M, Hu Z, Shi X, et al.: MAVS, cGAS, and endogenous retroviruses in T-independent B cell responses. Science. 2014; 346(6216): 1486–92. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nFogel LA, Yokoyama WM, French AR: Natural killer cells in human autoimmune disorders. Arthritis Res Ther. 2013; 15(4): 216. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRusakiewicz S, Nocturne G, Lazure T, et al.: NCR3/NKp30 contributes to pathogenesis in primary Sjogren's syndrome. Sci Transl Med. 2013; 5(195): 195ra96. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDudakov JA, Hanash AM, van den Brink MR, et al.: Interleukin-22: immunobiology and pathology. Annu Rev Immunol. 2015; 33: 747–85. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nCiccia F, Guggino G, Rizzo A, et al.: Potential involvement of IL-22 and IL-22-producing cells in the inflamed salivary glands of patients with Sjogren's syndrome. Ann Rheum Dis. 2012; 71(2): 295–301. PubMed Abstract | Publisher Full Text\n\nBarone F, Nayar S, Campos J, et al.: IL-22 regulates lymphoid chemokine production and assembly of tertiary lymphoid organs. Proc Natl Acad Sci U S A. 2015; 112(35): 11024–9. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDevauchelle-Pensec V, Mariette X, Jousse-Joulin S, et al.: Treatment of primary Sjögren syndrome with rituximab: a randomized trial. Ann Intern Med. 2014; 160(4): 233–42. PubMed Abstract | Publisher Full Text\n\nBowman S, Everett C, Bombardieri M, et al.: Preliminary Results of a Double-Blind Randomised Trial of Rituximab Anti-B-Cell Therapy in Patients with Primary Sjogrens Syndrome. [Abstract 11L]. Arthritis Rheumatol. 2015; 67(suppl 10). Reference Source\n\nde Vita S, Quartuccio L, Seror R, et al.: THU0392 Efficacy and Safety of Belimumab Given for 12 Months in Primary sjögren's Syndrome: The Beliss Open-Label Phase II Study. Ann Rheum Dis. 2015; 74(2): 338–339. [Poster Presentation]. Publisher Full Text\n\nCornec D, Costa S, Devauchelle-Pensec V, et al.: Blood and salivary-gland BAFF-driven B-cell hyperactivity is associated to rituximab inefficacy in primary Sjögren's syndrome. J Autoimmun. 2016; 67: 102–10. PubMed Abstract | Publisher Full Text\n\nWhang JA, Chang BY: Bruton's tyrosine kinase inhibitors for the treatment of rheumatoid arthritis. Drug Discov Today. 2014; 19(8): 1200–4. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMeiners PM, Vissink A, Kroese FG, et al.: Abatacept treatment reduces disease activity in early primary Sjögren's syndrome (open-label proof of concept ASAP study). Ann Rheum Dis. 2014; 73(7): 1393–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHu Y, Metz DP, Chung J, et al.: B7RP-1 blockade ameliorates autoimmunity through regulation of follicular helper T cells. J Immunol. 2009; 182(3): 1421–8. PubMed Abstract | Publisher Full Text\n\nSaadoun D, Rosenzwajg M, Joly F, et al.: Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis. N Engl J Med. 2011; 365(22): 2067–77. PubMed Abstract | Publisher Full Text\n\nLong SA, Rieck M, Sanda S, et al.: Rapamycin/IL-2 combination therapy in patients with type 1 diabetes augments Tregs yet transiently impairs β-cell function. Diabetes. 2012; 61(9): 2340–8. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSeror R, Bootsma H, Saraux A, et al.: Defining disease activity states and clinically meaningful improvement in primary Sjögren's syndrome with EULAR primary Sjögren's syndrome disease activity (ESSDAI) and patient-reported indexes (ESSPRI). Ann Rheum Dis. 2016; 75(2): 382–9. 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}
|
[
{
"id": "14447",
"date": "17 Jun 2016",
"name": "Charles Garrison Fathman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14451",
"date": "17 Jun 2016",
"name": "Xavier Mariette",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14453",
"date": "17 Jun 2016",
"name": "Robert Fox",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1412
|
https://f1000research.com/articles/5-1411/v1
|
17 Jun 16
|
{
"type": "Software Tool Article",
"title": "ddpcr: an R package and web application for analysis of droplet digital PCR data",
"authors": [
"Dean Attali",
"Roza Bidshahri",
"Charles Haynes",
"Jennifer Bryan",
"Dean Attali",
"Roza Bidshahri",
"Charles Haynes"
],
"abstract": "Droplet digital polymerase chain reaction (ddPCR) is a novel platform for exact quantification of DNA which holds great promise in clinical diagnostics. It is increasingly popular due to its digital nature, which provides more accurate quantification and higher sensitivity than traditional real-time PCR. However, clinical adoption has been slowed in part by the lack of software tools available for analyzing ddPCR data. Here, we present ddpcr – a new R package for ddPCR visualization and analysis. In addition, ddpcr includes a web application (powered by the Shiny R package) that allows users to analyze ddPCR data using an interactive graphical interface.",
"keywords": [
"droplet digital PCR",
"shiny",
"bioinformatics",
"personalized medicine",
"rpackage",
"gating",
"Gaussian mixture models",
"kernel density estimates"
],
"content": "Introduction\n\nDroplet digital polymerase chain reaction (ddPCR) accurately quantifies targeted nucleic acid sequences (templates) by randomly partitioning sample DNA into isolated droplets, such that most droplets contain at most one template. The template within each droplet is then amplified and detected in a sequence-specific manner using a hydrolysis probe. The counting of droplets emitting a sequence-specific fluorescent signal permits the number of copies of that sequence present in the sample to be quantified with excellent sensitivity and precision. Different templates, such as wild-type and mutant alleles, may be quantified by using a uniquely labeled probe against each. The most commonly used reporter dyes on the probes are FAM (fluorescein) and HEX™, with the end-point fluorescence amplitudes for the two dyes measured by analyzing each droplet with a two-channel fluorescence detector1.\n\nddPCR data readily lends itself to visualization as a two-dimensional scatter plot (Figure 1), in which the fluorescence amplitudes in both channels are plotted against each other for every droplet. In a ddPCR experiment designed to quantify two different templates, droplets ideally segregate into unique groups (clusters) that may include HEX-positive, FAM-positive, double-positive, and double-negative (empty) clusters2. For example, distinct FAM-positive, double-positive, and empty droplet clusters can be seen in Figure 5B. In practice, some droplets record an ambiguous set of fluorescent signals that fall between the distinct positive and negative populations. Such droplets are termed “rain” and can be observed between all clusters. By gating the droplets into groups based on their fluorescence signals, the exact number of template-positive droplets can be counted to provide exact quantification in a digital form.\n\nRaw ddPCR data from a two-channel ddPCR experiment (well F05 from the sample dataset).\n\n\nMotivation\n\nQuantification of template abundance from raw ddPCR data begins with assigning each droplet to a unique cluster or to rain. The QuantaSoft program (Bio-Rad, Hercules, CA) is designed to perform these assignments either via manual gating, with the usual disadvantages of subjectivity and non-reproducibility, or automatic gating. The algorithm used in the latter case is proprietary and can produce unsatisfactory results, especially when applied to ddPCR data obtained from formalin-fixed paraffin-embedded (FFPE) samples, as exemplified in Figure 5A.\n\nTwo third-party tools for automatic gating of ddPCR data have been described to date: ‘definetherain’ by Jones et al.3 and ’ddpcRquant’ by Trypsteen et al.4. However, both are limited to single-channel ddPCR data and are therefore not applicable to increasingly common two-channel experiments such as shown in Figure 1. Given the lack of tools for such analyses, users must currently resort to manual droplet gating.\n\n\nMethods\n\nTo improve automated droplet assignments as well as permit visualization of ddPCR datasets, we have developed ddpcr, an R package that can be used to explore, visualize, and analyze two-channel ddPCR data. The R language5 was chosen because it is open-source and cross-platform, which allows anyone to use it freely on any operating system. R is also a popular language in the field of computational biology, and is the main data analysis language for many scientists. To improve access and ease of use, we also implemented an interactive web application using Shiny6, through which one can run the analysis using a simple point-and-click interface.\n\nddpcr has been thoroughly tested using R versions 3.2.3 and 3.3.0 on both Windows 7 and Ubuntu 14.04.2 machines. However, the package is likely to run on any machine with a working installation of R.\n\nThe most important object in the ddpcr package is the ddpcr_plate object, or simply referred to as the \"plate object\". A plate object represents all the data for experiments conducted on a 96-well PCR plate. It gets created either by loading ddPCR input data files (see ‘Data import’) into a new plate object, or by loading an existing plate object that was previously saved to disk. A plate object contains all the information required to analyze the droplets within each well of a particular ddPCR plate. A plate object is both the input and output of all the core analysis functions.\n\nTo use the ddpcr package, it must first be installed and loaded.\n\n\n\nA very simple analysis workflow using a sample dataset can be performed using the following code, with the result of the code shown in Figure 2:\n\n\n\nddPCR data from well F05 of the sample dataset analyzed using ddpcr.\n\nWhile ddpcr contains dozens of functions, most analyses will follow a similar pattern: load ddPCR data into R using the new_plate() function, run the automated analysis using analyze(), and then explore the results using a variety of functions (Figure 3). The plot() function is used to visualize a dataset using ggplot27, while the plate_meta() and plate_data() functions return the dataset’s metadata and droplet grouping data as R data frames, respectively. The save_plate() function can be called at any time to save the current state of the dataset to disk in a format that can be loaded back into ddpcr.\n\nThe example code above uses a sample dataset, but in order to use new data, ddPCR data must be exported from QuantaSoft, as described in the next section. For more complex analysis or customizing the analysis parameters, see the full list of functions available by running ?ddpcr.\n\nBasic workflow for analyzing ddPCR data using the ddpcr package.\n\nBefore beginning analysis on a novel dataset, the first step is to import the ddPCR droplet fluorescence data into R. The raw data obtained from the fluorescence detector is encoded in a proprietary format that cannot be read by any software other than QuantaSoft, so the data must first be opened in QuantaSoft and exported into an accessible file format. QuantaSoft offers an option to export the droplet event data as a set of CSV (comma-separated values) files, as well as an option to export a metadata file that contains information on each well (Supplementary Figure 1 and Supplementary Figure 2). These CSV files are used as the input to ddpcr.\n\nThe analysis automatically gates droplets into unique clusters using kernel density estimation and Gaussian mixture models applied to the droplet fluorescence amplitudes. The full algorithm is explained in detail in a package vignette. The main analysis steps are:\n\nIdentify and exclude wells with a failed ddPCR reaction.\n\nIdentify and exclude outlier droplets, defined as those exhibiting a set of fluorescence amplitude signals characteristic of an error in the fluorescence readout.\n\nIdentify and exclude empty droplets — those displaying a set of signals indicative of complete absence of DNA template.\n\nCalculate the starting concentration of each template in the sample, defined as the number of copies per microlitre of input.\n\nAssign droplets into clusters by gating the droplets based on their fluorescence amplitudes. QuantaSoft’s automatic gating does not account for rain droplets and therefore can produce inaccurate results when the density of rain falls above a threshold. The gating algorithm in ddpcr accounts for rain and is therefore better able to distinguish clusters in clinical samples, such as FFPE samples, for which significant rain is often observed. Manual gating is also available in ddpcr to permit secondary verification of results.\n\nCount the number of droplets in each cluster.\n\nPlate objects are lists. Every S3 object in R has a base type upon which it is built. The plate object is implemented as an S3 object of class ddpcr_plate with the R list as its base type. Using a list allows for an easy way to bundle together the several different R objects describing a plate into one. All information required to analyze a plate is part of the plate object. Every plate object contains a set of nine elements that together fully describe and reproduce the current state of the dataset: plate_data, plate_meta, name, params, status, clusters, steps, dirty, version.\n\nUsing S3 to override base generic functions. Since the plate object is an S3 object, it can benefit from the use of generic functions. There are three common generic functions that the plate object implements: print(), plot(), and subset(). The print() method does not take any extra arguments and is used to print a summary of a plate object in a visually appealing way to the console. It gives an overview of the most important parameters of the plate such as its name and size. The plot() method generates a scatter plot of every well in the dataset and can be highly customizable using the many arguments it supports. While the base plot() method in R uses base R graphics, the plot() method for ddpcr_plate objects uses the ggplot2 package7. The subset() generic is overridden by a method that is used to retain only a subset of wells from a larger plate.\n\nPlate types. A ddPCR assay can be characterized by the droplet populations that are expected to arise after amplification. For example, in a (FAM+)/(FAM+HEX+) assay (such as Figure 1) it is expected that most of the non-empty droplets will either be FAM+HEX+ or FAM+, but not HEX+. Similarly, a (HEX+)/(FAM+HEX+) assay means that there are expected to be no droplets that are only FAM+. To describe these two types of assays, we define the term \"PN/PP\" (positive-negative/positive-positive). This name is a reflection of the expected populations of non-empty droplets: one population of singly-positive droplets (such as HEX+ or FAM+), and one population of double-positive droplets.\n\nThis characterization of a ddPCR experiment defines the plate type of a plate object, and it determines what type of analysis to run on the data. The default and most basic plate type is ddpcr_plate, which can be used for any ddPCR dataset. Running the analysis on a plate of this type will perform the first few analysis steps of identifying failed wells, outlier droplets, and empty droplets, but will not carry out the automated gating. Since in PN/PP-type experiments there is a rough expectation of where the droplets should be, automated gating can ensue on plates of that type.\n\nUsing S3 to support inheritance Inheritance means that every plate type has a parent plate type from which it inherits all its features, while specific behaviour can be added or modified. In ddpcr, transitive inheritance is implemented, which means that features are inherited from all ancestors rather than only the most immediate one. Multiple inheritance is not supported, meaning that each plate object can only have one parent.\n\nThe notion of inheritance is an important part of the ddpcr package, as it allows ddPCR data from different assay types to share many properties. For example, PN/PP assays are first treated using the analysis steps common to all ddPCR experiments, and then gated with an assay-specific step, so PN/PP assays can be thought of as inheriting the analysis from general ddPCR assays. Furthermore, the two types of PN/PP assays share many similarities, so they both inherit from a common PNPP plate type. Another benefit of inheritance in ddpcr is that it allows users to easily extend the functionality of the package by adding custom ddPCR plate types to gate different types of experiments. More information, including a fully worked example, on how to add a new plate type can be found in the package vignette (see ‘Software availability’).\n\nThe ddpcr package includes a web application that allows users to perform an analysis of ddPCR data in an interactive visual environment. The web application, written using the Shiny package v0.116, implements most of the features available in the ddpcr package and makes them accessible via a simple point-and-click interface. The Shiny application can be a useful tool for persons not comfortable with R programming or simply as a more convenient way to perform an analysis. However, since the web application only supports a curated subset of the ddpcr functions, it is not as powerful as using the command-line interface.\n\nThe ddpcr Shiny application includes four main tabs that mimic the natural flow of a ddPCR analysis (Figure 4): upload a dataset, configure analysis parameters, analyze the plate, and explore the results. At any point during the session, the current plate object can be downloaded and saved, and can be loaded into either the R command-line or the web application at a later time to continue the analysis.\n\nThe application is freely available online at http://daattali.com/shiny/ddpcr and is hosted on a server located in San Francisco, California. All data that is uploaded to the application is deleted when a user session ends, and none of the data is stored permanently. However, some users may prefer to run the application locally, which can be done using the ddpcr::launch() function.\n\nScreenshot from the ddpcr web application during an analysis of the sample ddPCR dataset.\n\n\nUse case\n\nWe have applied ddpcr to data (Dataset 1) from a novel ddPCR assay against somatic point mutations in the BRAF-V600 codon that was applied to FFPE specimens from a cohort of colorectal cancer (CRC) patients8. V600 mutations are observed in approximately 10% of colorectal tumours9 and their detection in CRC patients helps determine disease prognosis and treatment regimen. Through its droplet gating algorithm, ddpcr accurately identified droplet clusters and the number of droplets within each to provide the information needed to compute the frequency of mutated BRAF genes (Supplementary Figure 3).\n\nTo assess the accuracy of results from ddpcr, we compared BRAF-V600 mutation frequencies determined from the output of ddpcr with results obtained by two independent methods. V600 mutation frequencies computed from automated ddpcr results were within 3% of those obtained by manual analysis of the ddPCR data by an experienced operator (Supplementary Figure 4 and Supplementary Table 1). In addition, the BRAF-V600 status for each sample in the entire cohort was classified as mutant or wild-type by a certified pathologist using an immunohistochemical staining assay8. We obtained complete agreement between the pathologist’s binary classification of BRAF status and that determined using ddpcr.\n\nWe also analyzed the same dataset using QuantaSoft version 1.6.6. FAM-positive and double-positive droplets were not recognized as distinct clusters in 9 out of the 16 mutant-positive BRAF samples (Figure 5A).\n\nComparison between droplet gating in (A) QuantaSoft and (B) ddpcr. Both tools analyzed the same ddPCR experiment (well F05) from an assay designed to quantify wild-type (double-positive) and mutant (FAM-positive) alleles of the BRAF gene. (A) QuantaSoft failed to assign the double-positive and FAM-positive droplets into unique clusters, instead assigning all droplets recording a high FAM signal to a single cluster; (B) ddpcr assigned droplets into one of three uniquely identified clusters (double-positive (green), FAM-positive (orange), and empty (black)), or rain (blue).\n\n\nDiscussion\n\nWe present ddpcr, an R package that allows users to analyze ddPCR data and explore the results, both programmatically using R and via an interactive web application. To demonstrate clinical utility, a case study performed on a cohort of CRC patients showed that BRAF-V600 mutation frequencies determined using ddpcr are verified using two independent methods. The analysis runtime was 17 seconds, observed on a 64-bit Ubuntu 14.04.2 machine with 512MB of RAM and a single core Intel(R) Xeon(R) CPU E5-2630 at 2.30GHz. The package documentation includes details on extending the package, explanations of the algorithms used, and a walkthrough of a fully worked example.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw ddPCR data from application of the ddPCR assay against BRAF-V600 mutations, 10.5256/f1000research.9022.d12603210\n\nF1000Research: Dataset 2. The set of exported CSV files of the data presented in Dataset 1., 10.5256/f1000research.9022.d12603311\n\nDataset 1 is also available as a sample dataset within the ddpcr package. To access the data via the web application, select the tab Use sample dataset, choose Large dataset, and then click Load data. To access the data in R, run the following command to store the dataset as a plate object: my_data <- ddpcr::sample_plate(\"large\").\n\n\nSoftware availability\n\nSoftware available from: http://cran.r-project.org/package=ddpcr or https://github.com/daattali/ddpcr\n\nThe free web tool can be accessed online at: (http://daattali.com/shiny/ddpcr); or run locally via the ddpcr package with the command ddpcr::launch().\n\nLatest source code: https://github.com/daattali/ddpcr\n\nArchived source code at time of publication: https://dx.doi.org/10.6084/m9.figshare.342372512\n\nLicense: MIT",
"appendix": "Author contributions\n\n\n\nDA wrote the code and produced the figures. RB ran the ddPCR experiments. JB provided ideas and feedback for analysis algorithm. CH provided ideas and feedback for the functionality of the ddpcr package. DA wrote the manuscript with feedback from JB, RB, and CH. All authors approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nCH receives support as a Canada Research Chair. This work was further supported by the Canadian Institutes of Health Research (CIHR) Bioinformatics Training Program scholarship.\n\n\nAcknowledgments\n\nWe would like to thank Dr. Ryan Brinkman and his lab members for their time and advice.\n\n\nSupplementary material\n\nFigure 1. Exporting droplet fluorescence data from QuantaSoft.\n\nClick here to access the data\n\nFigure 2. Exporting plate metadata from QuantaSoft.\n\nClick here to access the data\n\nFigure 3. Automated ddpcr droplet gating results for raw assay output for the cohort of 32 CRC patient samples. The numbers show the calculated BRAF-V600 mutation frequency, defined as the ratio of FAM-positive droplets to the sum of FAM-positive and double-positive droplets. Background colours: green = sample is classified as wild-type, purple = sample is classified as mutant, grey = failed ddPCR run.\n\nClick here to access the data\n\nFigure 4. Comparison of the mutation frequency in each patient sample as calculated automatically by ddpcr vs that determined independently manually by an expert technician. The grey line represents the y = x line.\n\nClick here to access the data\n\nTable 1. Comparison of the mutation frequency in each patient sample as calculated automatically by ddpcr vs that determined independently manually by an expert technician.\n\nClick here to access the data\n\n\nReferences\n\nHindson BJ, Ness KD, Masquelier DA, et al.: High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem. 2011; 83(22): 8604–8610. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBizouarn F: Introduction to digital PCR. Methods Mol Biol. 2014; 1160: 27–41. PubMed Abstract | Publisher Full Text\n\nJones M, Williams J, Gärtner K, et al.: Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, ‘definetherain’. J Virol Methods. 2014; 202(100): 46–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTrypsteen W, Vynck M, De Neve J, et al.: ddpcRquant: threshold determination for single channel droplet digital PCR experiments. Anal Bioanal Chem. 2015; 407(19): 5827–5834. PubMed Abstract | Publisher Full Text\n\nR Core Team: R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, 2016. Reference Source\n\nChang W, Cheng J, Allaire JJ, et al.: shiny: Web Application Framework for R. R package version 0.13.2. 2016. Reference Source\n\nWickham H: ggplot2: Elegant Graphics for Data Analysis. Use R. Springer-Verlag New York, 2009. Publisher Full Text\n\nBidshahri R, Attali D, Fakhfakh K, et al.: Quantitative Detection and Resolution of BRAF V600 Status in Colorectal Cancer Using Droplet Digital PCR and a Novel Wild-Type Negative Assay. J Mol Diagn. 2016; 18(2): 190–204. PubMed Abstract | Publisher Full Text\n\nTol J, Nagtegaal ID, Punt CJ: BRAF mutation in metastatic colorectal cancer. N Engl J Med. 2009; 361(1): 98–99. PubMed Abstract | Publisher Full Text\n\nAttali D, Bidshahri R, Haynes C, et al.: Dataset 1 in: ddpcr: an R package and web application for analysis of droplet digital PCR data. F1000Research. 2016. Data Source\n\nAttali D, Bidshahri R, Haynes C, et al.: Dataset 2 in: ddpcr: an R package and web application for analysis of droplet digital PCR data. F1000Research. 2016. Data Source\n\nAttali D: ddpcr-1.3.zip. figshare. 2016. Data Source"
}
|
[
{
"id": "14448",
"date": "04 Jul 2016",
"name": "Timothy J. Triche Jr",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 ddpcr software is extensively documented and works as described. There are some minor changes that might be relevant (e.g. in ddpcr v1.4, the plot contains a percentage estimate in the lower right hand quadrant which is not shown in figure 1, and the X- and Y-axes are labelled slightly differently in the actual output from running the example. Overall, however, the package offers both a powerful tool for end users which advances the state of the art, and also a foundation for further algorithmic development. Cross-pollination between the flow cytometry community and ddpcr in the R ecosystem will likely lead to advances that would not otherwise have occurred.\n\nFigure 3 should be moved alongside the text in the paragraph \"Analysis algorithm\".\n\nFigure 5 is a powerful demonstration of the rationale for the library's creation and could perhaps stand to be moved up in the body of the paper. Standard practice is to provide a reason for the user to care about a method (e.g. figure 5), then describe the nuts and bolts of the implementation. There is a reason that this is standard practice.\n\nTime permitting, this reviewer has sought and obtained some much more challenging samples. In a revised or separate publication, it would be of interest to compare the software's performance on these ddPCR runs (where 1 in 10000 cells carries a mutation, but that 1 in 10000 is verified by multiple orthogonal sequencing methods) and determine whether the algorithmic flexibility of the ddpcr package would allow users to automate what is currently a highly technical process for detecting rare mutations. Unfortunately, the manufacturer has been unhelpful in converting this data to a usable format within the timeline of the review, so I cannot in good conscience delay it further.\nOutside of the scope of this paper, but relevant to the introduction, reproducibility and comparability would be much improved if the raw .QLP files could be parsed by R itself, making exchange and sharing of data far easier. Inquiries placed to Bio-Rad regarding the file format and the QuantaSoft package went unanswered, suggesting that (as with Illumina and their .IDAT format) the eventual solution will be to reverse engineer the format and brute-force the problem. It is unfortunate that some scientific instrument vendors appear to value imaginary profits over actual scientific merit, but such is life. Reverse engineering the file format is not a reasonable request here, but represents a future direction to further improve reproducibility of this important analytical technique. Existing tools rely upon ddpcr data provided in .CSV format; the first to elide this requirement (and that of a $5000 software package merely to review output) will be a notable step towards transparency for an increasingly powerful genotyping technique.\nIn conclusion, the ddpcr implementation and its extensive documentation (here in this paper and in the copious examples provided with the package) represent a solid foundation for further methodological improvements to an important assay platform.",
"responses": [
{
"c_id": "2147",
"date": "19 Aug 2016",
"name": "Dean Attali",
"role": "Author Response",
"response": "Thank you for the thorough and prompt review. I will address the comments once I get a review from the second reviewer (it's taking longer than expected to find someone!)"
}
]
},
{
"id": "15716",
"date": "14 Sep 2016",
"name": "Stephanie L. Hazlitt",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 tool article 'ddpcr: an R package and web application for analysis of droplet digital PCR data' is well-written and includes sufficient detail for the reader to assess the tool's construction, implementation and outputs. The ddpcr R package (v1.5) functions well and includes clearly-written, detailed vignettes to support the user and understand what is happening 'under the hood'. In addition, the source code is openly available on GitHub, which allows advanced R users to investigate details of the implementation. In general, we recommended more explicit links in the paper to supporting vignettes and the package README.\nIn addition to ddpcr being a useful tool for standard data science steps (get data, visualize data, analyze data), it also provides the ddPCR community with a well-documented, new analytical methodology for ddpcr clustering and hence opportunities for assessing robustness around clustering techniques in general in this field. This contribution could be made more clear in the Introduction and/or Motivation sections.\nFigure 5--referenced multiple times in the Introduction--could be moved alongside the text for the reader.\nThe Methods structure could be arranged to follow standard data science steps and Figure 3: (1) get data [raw, export, csv] (2) import data [new_plate()] (3)analyze data [analyze()]. There is no section on (4) visualize data [plot()], and the likely user cycle of analyze-visualize-change analysis parameters-analyze-visualize. With clustering, I am guessing this will be a common and important set of repeat steps. I would end the section with the example workflow code to reinforce the steps.\nThe Analysis sub-section might benefit from some supporting references for the selected analyses (e.g. kernel density estimation, Gaussian mixture models), url links to the ddpcr::analysis vignette in GitHub, and if the paper word-limit allowed, a bit more detail in the paper itself, especially for the 'assign droplets into clusters' step.\nThe examples show rain being identified in the FAM (empty vs filled; vertical direction), but not between FAM+ and FAM+HEX+ (mutant vs wildtype; horizontal direction). Is this because the algorithm doesn't allow for this, or because all of the droplets in that range could be classified as FAM+ or FAM+HEX+?\nAlso in the Analysis sub-section, a description of the next_step() function would be useful for those who want to understand each step in the process. A potential enhancement to the package would be a plot function/method for each step that visually displays the results of each step in the analysis.\nIt might be useful to synchronize the language used to describe the clusters with the attribute language in output objects in the package. For example, in the paper and vignettes clusters are \"double positive, FAM+, HEX+, or double negative\". In clusters() the clusters are POSITIVE NEGATIVE RAIN EMPTY etc. The plate_data function outputs clusters as numbers 1 through 7. The plate_meta function outputs clusters as mutant_num and wildtype_num. With a bit of sleuthing it is clear you can define the resulting cluster names with new_plate(), which is useful but may not be intuitive for many users.\nOther minor suggestions for potential enhancements after a test drive of ddpcr: - Plotting:\n\n- an addition of a Legend for the plot() would be a very useful\n\n- show_grid_labels = TRUE as default?\n\n- visually distinguish rain from empty? Figure 5 in the paper shows this, but the plot\n\ngenerated by running the code in the 'Workflow' section does not. - The plate_meta data.frame in a ddpcr_plate object could be a tibble for better printing - The plate_meta data.frame (after analysis) could also display the rain count",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1411
|
https://f1000research.com/articles/5-1409/v1
|
17 Jun 16
|
{
"type": "Review",
"title": "The changing faces of cholangitis",
"authors": [
"Sum P. Lee",
"Joseph R. Roberts",
"Rahul Kuver",
"Sum P. Lee",
"Joseph R. Roberts"
],
"abstract": "A variety of diseases are included under the umbrella term ‘cholangitis’, including hepatobiliary diseases with an autoimmune pathogenesis (such as primary sclerosing cholangitis, primary biliary cholangitis, and IgG4-associated sclerosing cholangitis) and disease processes associated with intraductal stones and infectious etiologies (such as ascending bacterial cholangitis, recurrent pyogenic cholangitis, and liver fluke-associated cholangitis). Recent advances in the pathophysiologic bases of these disorders, particularly with respect to the autoimmune variety, are allowing improved diagnosis and prognostication as well as providing the opportunity to refine and re-imagine treatment modalities. The aim of this review is to highlight selected advances in cholangitis research that point to novel insights into the pathophysiology, diagnosis, and treatment of this diverse array of disorders.",
"keywords": [
"cholangitis",
"primary sclerosing cholangitis",
"primary biliary cholangitis",
"IgG4-associated sclerosing cholangitis",
"cholangitis research"
],
"content": "Introduction\n\nThe term ‘cholangitis’ taken at face value means inflammation in the biliary system. This simple terminology is deceptive, however, as it encompasses a wide variety of diseases. These include liver fluke-associated cholangitis, ascending bacterial cholangitis associated with choledocholithiasis, recurrent pyogenic cholangitis, IgG4-associated cholangitis (IAC, also referred to as IgG4-related sclerosing cholangitis [ISC]), and primary sclerosing cholangitis (PSC). Recently, the designation for primary biliary cirrhosis (PBC) was changed to primary biliary cholangitis (conveniently also abbreviated to PBC)1, bringing yet another autoimmune disorder that affects the hepatobiliary system under the cholangitis umbrella. The term cholangiopathy has also been used to categorize several of these disorders, as the disease locus is the cholangiocyte or biliary epithelial cell2. The aim of this review is to summarize and put into perspective selected advances reported within the last 3 years that provide novel insights in our understanding of the pathophysiology, diagnosis, and treatment of these disorders.\n\n\nPathophysiology: new faces in the crowd\n\nPSC and PBC are autoimmune disorders, and a central tenet in the pathophysiology of these disorders is the complex interactions between cholangiocytes and cells in the innate and adaptive immune systems. Recent studies point to a central role for natural killer T (NKT) cells in the pathophysiology of these disorders3. The ability of cholangiocytes to express antigen-presenting molecules is established, but they were not previously known to activate NKT cells, a cell type abundant in the murine liver and also found to a lesser degree in the human liver. NKT cells respond to antigens presented with the major histocompatibility complex (MHC) class I-like molecule CD1d. Cultured human cholangiocytes and cholangiocarcinoma cells presented exogenous CD1d-restricted antigens to invariant NKT cell clones. Cd1d expression was downregulated in the biliary epithelium of patients with late PSC and PBC compared to healthy controls. This demonstrates that NKT cells can be activated by biliary epithelium and thereby regulate inflammation, which may be pathophysiologically relevant for PSC and PBC4. NKT cells also regulate T cell responses in PBC both during the initiation of disease as well as in the chronic phase5. A high ratio of NKT cells to cholangiocytes led to cytotoxicity of autologous cholangiocytes. At a low NKT/cholangiocyte ratio, cholangiocytes were not lysed, but interferon gamma (IFNγ) production was induced. This facilitated the expression of MHC class I and II molecules on cholangiocytes and afforded protection from lysis upon subsequent exposure to autoreactive T cells. Therefore, NKT cell-mediated innate immune responses are critical at the initial stage of PBC and also maintain the chronic cytopathic effect of autoantigen-specific T cells5. Invariant NKT cell-deficient mice have decreased portal inflammation and reduced anti-mitochondrial antibody responses6. These studies point to an important role for the innate immune system in general, and NKT cells in particular, in disease initiation and progression in both PBC and PSC.\n\nCD14 receptor signaling is another component of the innate immune system, and this receptor is constitutively expressed in the majority of innate immune cells. A recent report implicates this pathway in the pathogenesis of intrahepatic biliary strictures. Bile duct strictures are a hallmark of PSC and are a major cause of clinical consequences, causing biliary obstruction with ascending cholangitis and setting the stage for cholangiocarcinoma. CD14 receptor signaling was linked to dominant stricture formation in PSC patients in association with a specific genotype7.\n\nThe gut microbiome is a key component in the maintenance of health, and alterations in this meta-organ are increasingly being linked to the pathophysiologic basis for a wide variety of diseases8. Such alterations are particularly relevant for autoimmune conditions, including PSC. Approximately 75% of patients with PSC also have inflammatory bowel disease (IBD), predominantly ulcerative colitis (UC). This intersection of IBD with PSC has been the focus of studies to elucidate pathophysiologic mechanisms that could shed light on both diseases. Potential mechanisms for this close association between PSC and IBD include alterations in gut flora leading to changes in immune responses that could affect biliary epithelia and alterations in bile acid composition and signaling (e.g. via farnesoid X receptor [FXR], TGR5, and/or FGF19-mediated pathways) by gut microbial populations that could have downstream effects on biliary epithelia.\n\nMurine studies point to a role for the gut microbiome in biliary epithelial homeostasis. In the mdr2 (-/-) model of PSC, absence of the gut microbiota exacerbated hepatobiliary disease as shown by increased cholangiocyte senescence and more severe phenotypic features of PSC. Ursodeoxycholic acid (UDCA) abrogated cholangiocyte senescence9,10. These results demonstrate that the gut microbiota confers protection against injury to the biliary epithelium.\n\nRecent reports are beginning to shed light on microbial diversity with respect to patients with PSC (with and without IBD) in comparison to healthy controls. In one recent study, the gut microbial profile in patients with PSC was distinct from UC patients without biliary disease and healthy controls but similar in PSC patients with IBD11. PSC patients had reduced bacterial diversity compared to healthy controls. The Veillonella genus, which is also associated with other chronic inflammatory and fibrotic conditions, was more abundant in patients with PSC11. In another study, the mucosa-associated microbiota of patients with PSC was characterized by increased numbers of bacteria belonging to the Blautia and Barnesiellaceae genera and by major shifts in operational taxa units within the Clostridiales order12. In contrast, another group reported no strong PSC-specific microbial associations in UC patients13. While these studies are limited by small sample size and methodologic shortcomings, we anticipate future studies that will expand our understanding of the role of the gut microbiome in PSC and IBD. Two recent reviews highlight the current understanding of the gut microbiota with respect to hepatobiliary health and disease14,15.\n\nExosomes are secreted nanoparticles originating from endocytic vesicles that mediate cell-to-cell communication. Exosomes isolated from PBC patients and healthy controls showed effects on co-stimulatory molecule expression and cytokine production in mononuclear cells. There were differences in microRNA expression in circulating exosomes in patients with PBC compared to exosomes from healthy controls. These exosomes altered co-stimulatory molecule expression on antigen-presenting cells. These results suggest that aberrant exosomes in PBC selectively induce the expression of co-stimulatory molecules in different subsets of antigen-presenting cells and could contribute to the pathogenesis of PBC16.\n\n\nDiagnosis: effacing uncertainty\n\nDefining the relationship between PSC and IBD has implications that affect the clinical management of patients who carry either diagnosis. New evidence suggests that PSC associated with IBD is a distinct clinical entity compared to IBD without PSC. Disease-specific patterns at shared genetic loci showed strong co-morbidity between PSC and IBD, suggesting that PSC/IBD is a disease that is unique from IBD17. Patients with IBD and PSC have a markedly higher risk for the development of colorectal neoplasia than patients with IBD alone. UC rather than Crohn’s disease (CD) is associated with this higher risk in PSC patients18. The prevalence of PSC with CD is rare and carries a more benign prognosis as compared to patients with PSC with UC or without IBD. Approximately 25% of patients with concomitant PSC/CD had small duct PSC. In large duct PSC/CD, the liver disease was less aggressive and had a more favorable outcome19. Prolonged duration of IBD is associated with an increased risk of cholangiocarcinoma in patients with PSC/IBD, and colectomy does not modify this risk20. Screening for biliary malignancy with ERCP and brush cytology showed that even in asymptomatic PSC patients, 43% had advanced disease and 7% presented with suspicious or malignant brush cytology at first endoscopic retrograde cholangiography21. These studies highlight the ongoing efforts to delineate the interface between PSC and IBD.\n\nCholangitis is one of the common organ manifestations of IgG4-related disease; approximately 60% of patients with this systemic condition have changes consistent with sclerosing cholangitis in the proximal and/or distal bile ducts. ISC can result in hilar and even intrahepatic bile duct stenoses that can be difficult to differentiate from PSC and cholangiocarcinoma22. To further complicate matters, evidence is emerging that there is shifting of the diagnostic boundaries among ISC, biliary tract malignancy, and PSC23. Differential diagnosis between ISC and cholangiocarcinoma can be facilitated by a multi-modal diagnostic approach that includes the use of tumor markers, serum IgG4 levels, and other organ involvement. Differentiation between ISC and PSC can be improved by IgG4+ BCR clones and IgG4/IgG RNA ratio24. Response to steroid therapy can also help delineate these conditions in difficult cases25. However, a case report of extensive metastatic cholangiocarcinoma that was misdiagnosed as ISC illustrates these diagnostic limitations26. To further cloud the picture, PSC patients can have elevated IgG4 levels in serum, which is linked to specific human leukocyte antigen (HLA) haplotypes. Patients with the highest levels of IgG4 had significantly lower frequency of the strongest PSC risk factor, HLA-B*08, than patients without elevated IgG4. HLA genotype therefore might affect the serum concentration of IgG4, and increased IgG4 might be a marker of a distinct phenotype of PSC27. ISC and autoimmune pancreatitis have also been associated with extra-pancreatic organ failure and malignancy, requiring careful surveillance of these patients28. These studies highlight the ongoing clinical challenges regarding the diagnosis and management of patients when clinical features of ISC, PSC, and cholangiocarcinoma overlap.\n\nSerum and biliary biomarkers for early identification of PSC and cholangiocarcinoma are the subject of ongoing studies29. MicroRNAs have shown promise, with miR-1281, miR-126, miR-26a, miR-30b, and miR-122 showing significant differences between patients with PSC and patients with cholangiocarcinoma. In addition, these microRNAs were significantly lower in healthy individuals30. In bile, levels of miR-412, miR-640, miR-157, and miR-189 were significantly different between patients with PSC and patients with PSC/cholangiocarcinoma31. Another potential biomarker that is elevated in PSC is Pr3-ANCA; it can distinguish PSC from autoimmune hepatitis and PBC32. PSC can also be distinguished by elevated levels of biomarkers of apoptosis as opposed to biomarkers of necrosis33. Similarly, a panel of serum microRNAs was reported to be a more sensitive and specific biomarker for PBC than alkaline phosphatase or anti-nuclear antibodies34. Serum cell death biomarkers have also been found to predict liver fibrosis and poor prognosis in patients with PBC35.\n\nTraditional serum biomarkers continue to be refined. For example, normalization of alkaline phosphatase is a biomarker of improved survival in PSC and decreased likelihood of requiring orthotopic liver transplantation36. Reduction in alkaline phosphatase is also associated with longer survival in PSC independent of the presence of dominant strictures37. The serum biomarker CA 19-9 is associated with inflammation but not biliary obstruction in PSC patients38. Serum and urine bile acid and carnitine profiles may also be potential biomarkers in PBC39. The enhanced liver fibrosis score, which is a serum-based test of hyaluronic acid, TIMP-1, and propeptide of type III procollagen, has been shown to predict transplant-free survival in PSC40. Serum alkaline phosphatase and bilirubin levels are surrogate markers of outcomes in PBC41. Therefore, traditional and newer biomarkers continue to be developed and refined for use as tools for earlier and more specific diagnosis, as well as for helping with prognostication.\n\nEndoscopic technology continues to improve, allowing greater accuracy in the diagnosis of indeterminate biliary strictures, a challenge in the evaluation of dominant strictures in PSC. Cholangioscopy, using more sophisticated technological platforms, continues to evolve42. Cholangioscopes, confocal endoscopy, endoscopic ultrasound, and magnetic resonance-based imaging techniques provide ever more detailed information regarding strictures and other pathologic lesions in the biliary system. An example is the use of digital single operator cholangioscopy, which showed 85% sensitivity and 100% specificity for the diagnosis of malignancy arising within biliary strictures43.\n\n\nNew treatments: facing a bright future\n\nFor decades, the mainstay of treatment for PBC has been UDCA, which is non-toxic and widely available and has proven efficacy. However, up to 40% of patients with PBC will have an inadequate response to UDCA. FXR is a nuclear hormone receptor that is central to bile acid homeostasis in the hepatobiliary-gut axis. FXR activation has been associated with anti-cholestatic, anti-inflammatory, and anti-fibrotic effects. Obeticholic acid (OCA) is a potent FXR ligand that is being studied for the treatment of a variety of disorders. A double-blind study to assess the efficacy of OCA in PBC patients was recently reported; 165 patients on a stable dose of UDCA and with elevated alkaline phosphatase levels and no other immunosuppression or immunomodulatory therapy were randomized to escalating doses of OCA or placebo for 3 months. There was a decrease in alkaline phosphatase levels, with the majority of the effect seen after 1 month, suggesting that OCA may be a useful adjunct therapy to patients with PBC on UDCA with persistent biochemical abnormalities44. A dose-dependent side effect of OCA is pruritus, which is a common symptom in PBC patients. It is important to be cautious and introspective when considering OCA use. A long-term benefit has yet to be demonstrated. In addition, adverse serum lipid profiles and outcome may also severely limit its use.\n\nSeveral comprehensive reviews summarize the plethora of promising new therapies that are being tested in patients with PBC45–47. These novel approaches to treatment are based on insights into the cellular and molecular mechanisms involved in all stages of PBC, from the initiating and early immunologically mediated cholangiocyte injury to the damaging and disease-sustaining effects of cholestasis, leading ultimately to fibrosis and the development of cirrhosis. Therapies at various points in development include budesonide, fibrates, antivirals, B cell depletion with the anti-CD20 monoclonal antibody rituximab, anti-interleukin 12/interleukin 23 molecules such as ustekinumab, FGF-19 analogues, apical sodium-dependent bile acid transporter (ASBT) inhibitors, and TGR5 agonists, among others47. This wide array of treatment modalities, many of which are based on novel insights into the physiology of bile acid signaling pathways, nuclear hormone receptor signaling, and immunologic targets, are a testament to the advances in understanding of the cellular and molecular basis of PBC that have been decades in the making. Results of these studies are eagerly awaited. Proof of efficacy of one or more of these agents could dramatically alter the clinical management of patients with PBC who do not respond to UDCA.\n\nA similarly impressive array of novel therapeutics is under investigation for the medical management of PSC. Unlike PBC, the efficacy of UDCA remains unproven, with studies showing improvement in liver biochemistry profiles but no survival benefit. Furthermore, higher doses of UDCA are associated with adverse outcomes (reviewed in 48, with references therein). Due to this gap, there is great interest in developing effective therapies for this disease. Pre-clinical and clinical studies are ongoing and were recently reviewed49. Promising agents include nuclear hormone receptor agonists such as OCA for FXR, fibrates for PPARalpha, curcumin for PPARgamma, vitamin D for the vitamin D receptor, and all-trans retinoic acid for RAR/RXR. Another promising agent is the side-chain shortened derivative of UDCA, 24-norursodeoxycholic acid (norUDCA)48. Finally, agonists for TGR5 and for FGF-19 as well as inhibitors of ASBT are also candidates for drug development48. These drugs will change the management and outlook of PSC if shown to be effective in clinical trials.\n\n\nSummary\n\nThis review highlights the many ways in which our understanding of the pathophysiology, diagnosis, and management of the various diseases that are collectively labeled as cholangitis are evolving. We have chosen to highlight selected recently published advances that demonstrate novel insights, the majority of which involve the cholangitides with an autoimmune pathogenesis. The field of cholangitis studies is entering an unprecedented era of new advances that promise to translate into more effective prevention and management of this pathophysiologically fascinating and clinically challenging family of disorders. From novel insights regarding the role of the innate and adaptive immune systems to the not-unrelated role of the gut microbiome, our understanding of how these diseases develop and progress is expanding. Biomarker development with microRNAs and technological advances in endoscopy and imaging promise to provide earlier and more accurate diagnoses. In the therapeutic realm, the pipeline is full, and all stakeholders—patients and their families; basic, translational, and clinical investigators; and clinicians, among others—should feel cautiously optimistic about more effective treatments and improved outcomes.",
"appendix": "Competing interests\n\n\n\nThe authors have no personal or financial competing interests to declare.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBeuers U, Gershwin ME, Gish RG, et al.: Changing nomenclature for PBC: from 'cirrhosis' to 'cholangitis'. Gastroenterology. 2015; 149(6): 1627–9. PubMed Abstract | Publisher Full Text\n\nLazaridis KN, LaRusso NF: The Cholangiopathies. Mayo Clin Proc. 2015; 90(6): 791–800. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSebode M, Schramm C: Natural killer T cells: novel players in biliary disease? Hepatology. 2015; 62(4): 999–1000. PubMed Abstract | Publisher Full Text\n\nSchrumpf E, Tan C, Karlsen TH, et al.: The biliary epithelium presents antigens to and activates natural killer T cells. Hepatology. 2015; 62(4): 1249–59. 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J Crohns Colitis. 2016; 10(3): 330–7. PubMed Abstract | Publisher Full Text\n\nTabibian JH, O'Hara SP, Lindor KD: Primary sclerosing cholangitis and the microbiota: current knowledge and perspectives on etiopathogenesis and emerging therapies. Scand J Gastroenterol. 2014; 49(8): 901–8. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nTabibian JH, Varghese C, LaRusso NF, et al.: The enteric microbiome in hepatobiliary health and disease. Liver Int. 2016; 36(4): 480–7. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nTomiyama T, Yang GX, Zhao M, et al.: The modulation of co-stimulatory molecules by circulating exosomes in primary biliary cirrhosis. Cell Mol Immunol. 2015. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nEllinghaus D, Jostins L, Spain SL, et al.: Analysis of five chronic inflammatory diseases identifies 27 new associations and highlights disease-specific patterns at shared loci. Nat Genet. 2016; 48(5): 510–8. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nZheng HH, Jiang XL: Increased risk of colorectal neoplasia in patients with primary sclerosing cholangitis and inflammatory bowel disease: a meta-analysis of 16 observational studies. Eur J Gastroenterol Hepatol. 2016; 28(4): 383–90. PubMed Abstract | F1000 Recommendation\n\nFevery J, van Steenbergen W, van Pelt J, et al.: Patients with large-duct primary sclerosing cholangitis and Crohn's disease have a better outcome than those with ulcerative colitis, or without IBD. Aliment Pharmacol Ther. 2016; 43(5): 612–20. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGulamhusein AF, Eaton JE, Tabibian JH, et al.: Duration of Inflammatory Bowel Disease Is Associated With Increased Risk of Cholangiocarcinoma in Patients With Primary Sclerosing Cholangitis and IBD. Am J Gastroenterol. 2016; 111(5): 705–11. 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PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHuggett MT, Culver EL, Kumar M, et al.: Type 1 autoimmune pancreatitis and IgG4-related sclerosing cholangitis is associated with extrapancreatic organ failure, malignancy, and mortality in a prospective UK cohort. Am J Gastroenterol. 2014; 109(10): 1675–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO'Hara SP, Gradilone SA, Masyuk TV, et al.: MicroRNAs in Cholangiopathies. Curr Pathobiol Rep. 2014; 2(3): 133–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVoigtlander T, Gupta SK, Thum S, et al.: MicroRNAs in Serum and Bile of Patients with Primary Sclerosing Cholangitis and/or Cholangiocarcinoma. PLoS One. 2015; 10(10): e0139305. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nGradilone SA, O'Hara SP, Masyuk TV, et al.: MicroRNAs and benign biliary tract diseases. Semin Liver Dis. 2015; 35(1): 26–35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStinton LM, Bentow C, Mahler M, et al.: PR3-ANCA: a promising biomarker in primary sclerosing cholangitis (PSC). PLoS One. 2014; 9(11): e112877. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMasuoka HC, Vuppalanchi R, Deppe R, et al.: Individuals with Primary Sclerosing Cholangitis Have Elevated Levels of Biomarkers for Apoptosis but Not Necrosis. Dig Dis Sci. 2015; 60(12): 3642–6. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nTan Y, Pan T, Ye Y, et al.: Serum microRNAs as potential biomarkers of primary biliary cirrhosis. PLoS One. 2014; 9(10): e111424. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSekiguchi T, Umemura T, Fujimori N, et al.: Serum cell death biomarkers for prediction of liver fibrosis and poor prognosis in primary biliary cirrhosis. PLoS One. 2015; 10(6): e0131658. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHilscher M, Enders FB, Carey EJ, et al.: Alkaline phosphatase normalization is a biomarker of improved survival in primary sclerosing cholangitis. Ann Hepatol. 2016; 15(2): 246–53. PubMed Abstract | F1000 Recommendation\n\nRupp C, Rössler A, Halibasic E, et al.: Reduction in alkaline phosphatase is associated with longer survival in primary sclerosing cholangitis, independent of dominant stenosis. Aliment Pharmacol Ther. 2014; 40(11–12): 1292–301. PubMed Abstract | Publisher Full Text\n\nWannhoff A, Rupp C, Friedrich K, et al.: Inflammation But Not Biliary Obstruction Is Associated With Carbohydrate Antigen 19-9 Levels in Patients With Primary Sclerosing Cholangitis. Clin Gastroenterol Hepatol. 2015; 13(13): 2372–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTang YM, Wang JP, Bao WM, et al.: Urine and serum metabolomic profiling reveals that bile acids and carnitine may be potential biomarkers of primary biliary cirrhosis. Int J Mol Med. 2015; 36(2): 377–85. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nVesterhus M, Hov JR, Holm A, et al.: Enhanced liver fibrosis score predicts transplant-free survival in primary sclerosing cholangitis. Hepatology. 2015; 62(1): 188–97. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLammers WJ, van Buuren HR, Hirschfield GM, et al.: Levels of alkaline phosphatase and bilirubin are surrogate end points of outcomes of patients with primary biliary cirrhosis: an international follow-up study. Gastroenterology. 2014; 147(6): 1338–49.e5; quiz e15. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGhersi S, Fuccio L, Bassi M, et al.: Current status of peroral cholangioscopy in biliary tract diseases. World J Gastrointest Endosc. 2015; 7(5): 510–7. PubMed Abstract | Free Full Text\n\nNavaneethan U, Hasan MK, Kommaraju K, et al.: Digital, single-operator cholangiopancreatoscopy in the diagnosis and management of pancreatobiliary disorders: a multicenter clinical experience (with video). Gastrointest Endosc. 2016; pii: S0016-5107(16)01051-8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHirschfield GM, Mason A, Luketic V, et al.: Efficacy of obeticholic acid in patients with primary biliary cirrhosis and inadequate response to ursodeoxycholic acid. Gastroenterology. 2015; 148(4): 751-61.e8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDyson JK, Hirschfield GM, Adams DH, et al.: Novel therapeutic targets in primary biliary cirrhosis. Nat Rev Gastroenterol Hepatol. 2015; 12(3): 147–58. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTabibian JH, Lindor KD: Primary biliary cirrhosis: safety and benefits of established and emerging therapies. Expert Opin Drug Saf. 2015; 14(9): 1435–44. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAli AH, Tabibian JH, Carey EJ, et al.: Emerging drugs for the treatment of Primary Biliary Cholangitis. Expert Opin Emerg Drugs. 2016; 21(1): 39–56. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHalilbasic E, Fuchs C, Hofer H, et al.: Therapy of Primary Sclerosing Cholangitis--Today and Tomorrow. Dig Dis. 2015; 33(Suppl 2): 149–63. PubMed Abstract | Publisher Full Text\n\nWilliamson KD, Chapman RW: New Therapeutic Strategies for Primary Sclerosing Cholangitis. Semin Liver Dis. 2016; 36(1): 5–14. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "14440",
"date": "17 Jun 2016",
"name": "Keith Lindor",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14441",
"date": "17 Jun 2016",
"name": "Albert Pares",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1409
|
https://f1000research.com/articles/5-1407/v1
|
17 Jun 16
|
{
"type": "Case Report",
"title": "Case Report: Kikuchi-Fujimoto disease: a diagnostic and therapeutic dilemma following pretransplant nephrectomy for a 2.35 Kg kidney",
"authors": [
"Arvind P. Ganpule",
"Jaspreet Singh Chabra",
"Abhishek G. Singh",
"Gopal R. Tak",
"Shailesh Soni",
"Ravindra Sabnis",
"Mahesh Desai",
"Jaspreet Singh Chabra",
"Abhishek G. Singh",
"Gopal R. Tak",
"Shailesh Soni",
"Ravindra Sabnis",
"Mahesh Desai"
],
"abstract": "Kikuchi-Fujimoto disease (KFD) is an extremely rare disease with a worldwide distribution and higher prevalence in Asians. It is a benign and self-limiting disorder, characterized by regional cervical lymphadenopathy accompanied with mild fever and night sweats. Lymph node histopathology is diagnostic and treating physicians should be aware of this entity as it may mimic other systemic diseases like systemic lupus erythematosus, tuberculosis, malignant lymphoma, and more rarely adenocarcinoma. Key features on lymph node biopsy are fragmentation, necrosis and karyorrhexis. Treatment includes symptomatic care, analgesics-antipyretics, corticosteroids and spontaneous recovery occurs in 1 to 4 months. We report a case of adult polycystic kidney disease (ADPKD) with end stage renal disease and episodes of fever and cervical lymphadenopathy. The infectious screen was negative and on extensive workup, the patient was found to have histiocytic-necrotizing lymphadenitis, which clinched the diagnosis of KFD.",
"keywords": [
"Kikuchi Fujimoto disease",
"Autosomal dominant polycystic kidney disease",
"Lymphadenopathy"
],
"content": "Introduction\n\nKikuchi-Fujimoto disease (KFD), or histiocytic necrotizing lymphadenitis, is a benign and self-limiting disease that mainly affects young women. Recognition of this condition is critical as it can mimic tuberculosis, lymphoma, or even adenocarcinoma. Awareness of this entity helps to prevent misdiagnosis and inappropriate treatment1.\n\nThe aim of this report is to review the authors' institutional experience of KFD in a patient with end stage renal disease being prospectively evaluated for renal allograft transplant. Diagnosis of KFD should be kept in mind in patients who present with fever and cervical lymphadenopathy. This presentation may not be a rarity in immunocompromised pre and post transplant patients. We report a case of KFD in a patient with adult polycystic kidney disease who underwent bilateral pre-transplant nephrectomy.\n\nKeywords: Kikuchi – Fujimoto disease (KFD), Autosomal dominant polycystic kidney disease (ADPKD), Lymphadenopathy\n\n\nCase history\n\nA 42 years old Asian male on evaluation of abdominal pain was diagnosed with adult polycystic kidney disease (ADPKD) in 2002. The patient’s mother died of renal failure due to ADPKD. Patient had progressive deterioration of renal function and developed chronic kidney disease (CKD) stage 5 in 2015. The patient was advised renal replacement therapy (RRT) and required thrice-weekly hemodialysis since September 2015. He was evaluated for renal transplant and planned for the same in May 2016. During pre-transplant evaluation he developed fever with chills and bilateral painful cervical lymphadenopathy in the posterior triangle of neck. Ultrasound of cervical region reported presence of multiple enlarged lymph nodes in the neck along internal jugular vein on both the sides; largest node on right side measured 21×7 mm in size and on the left side largest node measured 16×5 mm in size. Ultrasound examination of axilla and groin did not reveal lymphadenopathy.\n\nThe patient underwent right cervical lymph node biopsy. Histopathology was reported to be acute necrotizing lymphadenitis. This biopsy was negative for acid-fast bacilli (AFB) and fungal stains. In the meanwhile his complete infectious screen of blood, urine, sputum and stool cultures showed no growth. The patient was given broad-spectrum third generation cephalosporins for 14 days assuming the fever to be caused by unidentifiable bacterial infection.\n\nThe fever settled and the patient was planned for a bilateral pre-transplant nephrectomy in view of bilateral large size kidney and recurrent fever. Right laparoscopic Pre-transplant nephrectomy was done on 05 February 2016. This was followed by a pre-transplant left open nephrectomy on 16 February 2016. The left side was planned for an open nephrectomy in view of the large size of the kidney. The procedure was uneventful. Incidentally, this was one of the largest kidney specimen ever recorded in literature weighing 2.35 Kg (Figure 1a & 1b). The Guinness Book of World Records reports a kidney weighing 2.15 kg as world's largest kidney till now which was retrieved in Dhule, Maharashtra, India2.\n\nFigure 1a is showing length of the specimen in cm and Figure 1b shows weight of the specimen in Kg on a certified weighing scale.\n\nPostoperatively, the patient had continuous fever of 100 degree Fahrenheit with chills for 10–15 days; despite he receiving third generation broad-spectrum injectable antibiotics. As the patient had undergone a bilateral nephrectomy, the first diagnosis for pyrexia of unknown origin was postoperative surgical infection or intra-abdominal abscess formation. The imaging done included high-resolution computerized tomography (CT) chest (HRCT chest), CT abdomen and CT pelvis. These did not reveal any significant finding. His fever did not subside despite all the measures such as higher antibiotics, antipyretics and removal of all indwelling catheters. His indwelling catheter lines were cultured to rule out any infection, but did not grow any organism. An infectious disease specialist was consulted and he advised for a slide review of the cervical lymph node sample, which was done prior to nephrectomy.\n\nSlide review with the specialist suggested a necrotizing histiocytic lymphadenitis (Kikuchi Disease) (Figure 2a & 2b). Section revealed lymphoid architecture being replaced by necrotizing lesions, which are composed of karyorrhectic debris with fibrin deposits and collection of mononuclear cells. Plasma cells and neutrophils were scanty. Periodic acid-Schiff (PAS) stain was negative for fungal hyphae. Ziehl Neelsen (ZN) stain was negative for AFB with final diagnosis as necrotizing histiocytic lymphadenitis. Tests for bacteria/fungus/AFB were negative. Genexpert examination for tubercle bacilli (TB) was also negative. Immmunohistochemistry (IHC) examination for lymphoma was negative. Tests for systemic lupus erythematosus (SLE) were also negative. The key features which clinched the histological diagnosis were: karyorrhexis, fragmentation and necrosis.\n\nFigure 2a showing low power light microscopic view of lymphnode biopsy highlighting karyorrhexis and necrosis. Figure 2b showing a high power light microscopy images of cervical lymphnode biopsy demonstrating histiocytes.\n\nThe patient was started on non-steroidal anti-inflammatory drugs (NSAID), oral Prednisolone 10 mg twice a day for 15 days and continued on hemodialysis (HD). Patient responded well to this treatment measures. A team of an infection specialist, a nephrologist and a urologist managed this patient. The patient finally became afebrile and fit for renal transplantation after a treatment for 3 weeks. He underwent a living related renal transplant on 27th of April 2016. His postoperative course after renal transplantation was uneventful and achieved a nadir creatinine of 1.05 mg/dl.\n\n\nDiscussion\n\nKFD, or histiocytic necrotizing lymphadenitis, was originally reported in 1972 in Japan3. KFD is a rare disease, more commonly occurring in females of Asian origin during the third decade of life, although it has been reported between 19 months to 75 years of age. Association of KFD with HLA class II alleles (HLA-DPA1 and HLA-DPB1 especially) has been described in Asian KFD patients4.\n\nIncidence of KFD is higher in females with the most common presentation being cervical lymphadenopathy and with half of patients presenting fever with leucopenia5–7. Patients suspected to have this disease should be extensively examined and tested for tuberculosis, Epstein-Barr virus, cytomegalovirus, human immunodeficiency virus and SLE. Lymphoma can also be mimicked by this disease and has to be ruled out by immmunohistochemistry. Key histological features of KFD include single or focal areas within lymph node containing histiocytic cellular infiltrate with necrosis, with perinodal inflammation and occasional capsular invasion8.\n\nThe signs and symptoms of Kikuchi disease are fever, lymphadenopathy, skin rashes and headache. Rarely, hepato-splenomegaly and nervous system involvement resembling meningitis are seen. This disease is also associated with bouts of extreme fatigue, especially during latter parts of the day.\n\nDifferential diagnosis of KFD includes SLE, disseminated tuberculosis, lymphoma, sarcoidosis and viral lymphadenitis. Clinical findings sometimes may include positive results for IgM/IgG/IgA antibodies for respective diseases. In contrast to published reports of KFD, which showed a female predominance, in our case it occurred in a male patient.\n\n\nConclusion\n\nKFD, although rare, should be part of the differential diagnosis in patients presenting with fever and cervical lymphadenopathy in chronic kidney disease patients, especially in patients of Asian origin who presents a negative infectious screen.\n\nKey messages from our case report:\n\n1) High degree of suspicion is required for diagnosis of KFD. KFD is a self-limiting benign disorder characterized by lymphadenopathy, which resolves in a few weeks to months.\n\n2) The pillars for diagnosis for KFD include histological features of necrosis, fragmentation and karyorrhexis in lymph node biopsy.\n\n3) In immunosuppressed patients as in this case, the diagnostic dilemma remains and the differential diagnosis to be considered include systemic lupus erythematosus (SLE), disseminated tuberculosis, lymphoma, sarcoidosis, and viral lymphadenitis.\n\n4) Apart from the work up of pyrexia of unknown origin, an experienced histopathologist should do a slide review of cervical lymphnode biopsy.\n\n5) The management should be multimodal; as in this case it involved a nephrologist, an urologist and an infection disease specialist. Treatment is mostly supportive and steroids may be helpful in the treatment.\n\n\nPatient consent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\n1. Dr. Ganpule Arvind: Concept, manuscript editing.\n\n2. Dr. Singh Abhishek: Manuscript writing and editing.\n\n3. Dr. Gopal Tak: Manuscript writing.\n\n4. Dr. Chhabra Jaspreet: Manuscript editing.\n\n5. Dr. Sabnis Ravindra: Manuscript editing.\n\n6. Dr. Shailesh Soni: Histopathological description and images.\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\nRammohan A, Cherukuri SD, Manimaran AB, et al.: Kikuchi-Fujimoto disease: a sheep in wolf's clothing. J Otolaryngol Head Neck Surg. 2012; 41(3): 222–6. PubMed Abstract\n\nwww.guinnessworldrecords.com/world-records/largest-kidney.\n\nKikuchi M: Lymphadenitis showing focal reticulum cells hyperplasia with nuclear debris and phagocytosis: A clinicopathological study. Nippon Ketsueki Gakkai Zasshi. 1972; 35: 379–80. Reference Source\n\nTanaka T, Ohmori M, Yasunaga S, et al.: DNA typing of HLA class II genes (HLA-DR, -DQ and -DP) in Japanese patients with histiocytic necrotizing lymphadenitis (Kikuchi's disease). Tissue Antigens. 1999; 54(3): 246–53. PubMed Abstract | Publisher Full Text\n\nLopez C, Oliver M, Olavarria R, et al.: Kikuchi-Fujimoto necrotizing lymphadenitis associated with cutaneous lupus erythematosus: a case report. Am J Dermatopathol. 2000; 22(4): 328–33. PubMed Abstract | Publisher Full Text\n\nKaur S, Thami GP, Kanwar AJ: Kikuchi's disease, skin and systemic lupus erythematosus. Br J Dermatol. 2002; 146(1): 167–8. PubMed Abstract | Publisher Full Text\n\nNorris AH, Krasinskas AM, Salhany KE, et al.: Kikuchi-Fujimoto disease: a benign cause of fever and lymphadenopathy. Am J Med. 1996; 101(4): 401–5. PubMed Abstract | Publisher Full Text\n\nIoachim HL, Medeiros LJ editors: Ioachim's lymph node pathology. Lippincott Williams & Wilkins, 2009. Reference Source"
}
|
[
{
"id": "14541",
"date": "23 Jun 2016",
"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\nThe article is well written and indeed presents an extremely rare occurrence. I have little to add, I would recommend indexing.\nI would amend the bit about the Guinness World Record, Medicine is not a contest.",
"responses": []
},
{
"id": "14562",
"date": "24 Jun 2016",
"name": "Sanjay B. Kulkarni",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case report. KFD is a treatable but rare disorder. In this particular patient it was diagnosed due to a vigilant clinician and pathologist. KFD should be kept in differential diagnosis.\n\nI recommend that this article is indexed.",
"responses": []
}
] | 1
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https://f1000research.com/articles/5-1407
|
https://f1000research.com/articles/5-136/v1
|
04 Feb 16
|
{
"type": "Research Note",
"title": "GPR21 KO mice demonstrate no resistance to high fat diet induced obesity or improved glucose tolerance",
"authors": [
"Jinghong Wang",
"Zheng Pan",
"Helene Baribault",
"Danny Chui",
"Caroline Gundel",
"Murielle Véniant",
"Jinghong Wang",
"Zheng Pan",
"Helene Baribault",
"Danny Chui",
"Caroline Gundel"
],
"abstract": "Gpr21 KO mice generated with Gpr21 KO ES cells obtained from Deltagen showed improved glucose tolerance and insulin sensitivity when fed a high fat diet. Further mRNA expression analysis revealed changes in Rabgap1 levels and raised the possibility that Rabgap1 gene may have been modified. To assess this hypothesis a new Gpr21 KO mouse line using TALENS technology was generated. Gpr21 gene deletion was confirmed by PCR and Gpr21 and Rabgap1 mRNA expression levels were determined by RT-PCR. The newly generated Gpr21 KO mice when fed a normal or high fat diet chow did not maintain their improved metabolic phenotype. In conclusion, Rabgap1 disturbance mRNA expression levels may have contributed to the phenotype of the originally designed Gpr21 KO mice.",
"keywords": [
"GPCR",
"Rabgap1",
"Diabetes",
"Drug target",
"TALENS technology"
],
"content": "Introduction\n\nThe G-protein receptors (GPCRs) are the largest family of proteins targeted by drug discovery. GPCRs are crucial molecular sensors for many vital physiological processes. GPR21 is part of the GPCRs family and shares 71% identity to GPR52. It was identified along with GPR22 and GPR23 based on their homology to GPR20 (O’Dowd et al., 1997). Originally, GPR21 was detected in regions of the brain and later, several other tissues as spleen, brown fat, and macrophages, were reported to express high levels of GPR21 mRNA (Gardner et al., 2012; Osborn et al., 2012). The natural ligand of GPR21 remains unknown; however, constitutive activity of the GPR21 receptor has been observed when it was co-transfected with Gα15/16 proteins in HEK293 cells (Gardner et al., 2012; Xiao et al., 2008). Also, GPR21 has been reported to activate the Gq pathway on calcium-sensitive CHO cells (Bresnick et al., 2003).\n\nIn Gpr21 KO mice generated with Gpr21 KO ES cells obtained from Deltagen (Deltagen GPR21, Deltagen San Mateo, CA), Osborn et al. and Gardner et al. have reported that glucose tolerance and insulin sensitivity were improved when compared to their wildtype control mice (Gardner et al., 2012; Osborn et al., 2012). These Gpr21 KO mice were leaner than their wildtype littermate control (Osborn et al., 2012) and were resistant to diet-induced obesity (Gardner et al., 2012), making GPR21 a potential drug target candidate for the treatment of diabetes and obesity. Reduced inflammation and macrophage infiltration were also observed in the KO mice (Osborn et al., 2012).\n\nMouse Gpr21 gene is located on chromosome 2 within the intron of Rabgap1 gene, between exon 13 and 14 according to the UCSC GRCm38/mm10 assembly. Strbp gene is located on the opposite strand in the same region. Deltagen Gpr21 KO mice contain a deletion in the gene of exon one with the insertion of a 5.3 kb lacZ/Neo cassette. After considering the location of the insertion of the neo cassette, we hypothesized that the gene structures, the expression and physiological functions of Rabgap1 and Strbp may have been altered.\n\nIn brief, small RAB GTPases are essential for the coordination of vesicle budding, transport, and fusion of vesicles (Frasa et al., 2012). RAB proteins are activated by guanine nucleotide exchange factors (GEFs) and inactivated by RAB GTPase activating proteins (RABGAPs). The TBC (TRE2-BUB2-CDC16) domain facilitates the RAB GTP hydrolysis from the GTP-bound active form to the GDP-bound inactive form. However, the physiological function of RABGAP1 (TBC1D11) is less understood. It may be implicated in microtubule and Golgi dynamics during cell cycle and regulation of spindle checkpoint (Cuif et al., 1999; Miserey-Lenkei et al., 2006). STRBP (SPNR) is a microtubule-associated RNA-binding protein localized in developing spermatids and plays an important role in normal spermatogenesis and sperm function (Pires-daSilva et al., 2001). Strbp deficient mice are smaller, have neurological defects, a high premature mortality rate, show reduced fertility and mating drive as well as abnormal sperm motility.\n\nAfter further analysis of the Deltagen Gpr21 KO mice, we observed that Rabgap1 mRNA expression levels were modified. To assess if the metabolic phenotype observed in these KO mice (Gardner et al., 2012; Osborn et al., 2012) was solely related to knocking out the Gpr21 gene, we generated a new line of Gpr21 KO mice using the TALENS technology. We created a 29 bp deletion within the coding exon of Gpr21 (Gpr21 TAL 29bp), a location very close to the ATG, an out of frame mutation and an early termination of Gpr21. The phenotypic analysis of our new Gpr21 TAL 29bp KO mice showed no improvement of the previously observed metabolic parameters that were identified in Deltagen Gpr21 KO mice. The originally published improved metabolic phenotype of the Deltagen Gpr21 KO mice was not solely due to the deletion of the Gpr21 gene, Rabgap1 may have been implicated.\n\n\nMaterials and methods\n\nAll animal experiments were approved by the Institutional Animal Care and Use Committee of Amgen (animal protocol #2006-00010). Mice were housed in a pathogen-free facility with a 12 h light-dark cycle. Mice were allowed ad libitum access to water and food. They were fed with normal chow (Harlan 2920) before starting a high fat diet (Research Diets D12451, 45 kcal % fat).\n\nGpr21 KO mice were created using a pair of transcription activator-like effector nucleases (TALENs) from Life Technologies targeting exon 2 of mouse Gpr21 TALEN binding sites are underlined below with a 15 base pair spacer between the 2 sites.\n\n5 ’- TGAACTCCACCTGGGATGG TAATCAGAGCAGCCA TCCTTTCTGTCTTCTGGCA\n\nACTTGAGGTGGACCCTACC ATTAGTCTCGTCGGT AGGAAAGACAGAAGACCGT - 5’\n\nDesign, cloning and validation of the TALENs were performed by Life Technologies. Messenger RNA (provided from Life Technologies) for each of the TALENs were diluted in RNAse free microinjection buffer to a final concentration of 4.0 ng/µl for each TALEN (8.0 ng/µl total concentration). The TALENs were microinjected into the pronucleus of fertilized one-cell embryos (0.5 days post coitus) obtained from the mating of C57BL/6 (Taconic) males to superovulated C57BL/6 (Taconic) female mice. Microinjected eggs were transferred to pseudopregnant Swiss Webster recipients. Founder pups were screened for TALEN induced mutations in Gpr21 by sequencing across exon 2. Two founders, one with a 5 bp deletion and the other with a 29 bp deletion were expanded for further analysis.\n\nGenomic DNA was prepared from liver, BAT and spleen using DNeasy blood and tissue kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. PCR was carried out using the primers 5’-CAGCATGAAGTGAGAGCCAG-3’ and 5’-CAAGTAGCCCAGTGCCAGAAG-3’.\n\nFor the microarray analysis, mRNA was isolated from 6 animals for each group using Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA) and processed following the protocols described in section 2 (Eukaryotic Sample and Array Processing; 701024 rev 1) of the Affymetrix Technical Manual. Briefly, 5 µg total RNA was used to synthesize cDNA (10 pmol of T7-(dT)24 primer, and Superscript II (Invitrogen, Carlsbad, CA). Purified double-stranded cDNA (MinElute Reaction Cleanup Kit, Qiagen, Valencia, CA) was used to generate biotinylated cRNA using Bioarray HighYield RNA Transcript labeling Kit (Enzo Diagnostics, Farmingdale, NY) followed by purification with Qiagen RNeasy Mini kit and hybridization to the Affymetrix HT MG 430 PM array. Arrays were washed on a GeneChip Fluidic Station 450 (EukGE_WS2v4_450 protocol) and scanned using the Affymetrix GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA). Data analysis was conducted with R (version 2.15, http://r-project.org), Bioconductor (version 2.10, http://bioconductor.org/) and ArrayStudio (Omicsoft, version 8.0). Briefly, Affymetrix CEL files were normalized in Bioconductor using the GCRMA method. Differentially regulated genes were identified using a moderated t-test. False discovery rate adjusted P-values were calculated using the method of Benjamini & Hochberg (1995).\n\nTotal RNA was isolated using Qiagen midi RNA preparation kit (Qiagen, Valencia, CA). The total RNA concentration was determined with a Nanodrop (ThermoFisher Scientific, Wilmington, DE). QPCR was performed using 10 ng RNA per well, Taqman master mix (Applied Biosystems, Foster City, CA). Rabgap1 gene expression was assessed using Taqman probes from Applied Biosystems (Mm01327207_m1 and Mm01327199_m1). Gpr21 mRNA level was measured using the forward primer 5’-CACCTGGGATGGTAATCAGAG-3’, reverse primer 5’- TCACAATGATGTTGCCAGAAAT-3’ and probe 5’ FAM/TTCTGGCAC/Zen/TGGGCTACTTGGAAA/IABkFQ-3’ from Integrated DNA Technologies (Coralville, IA). Results were evaluated using the ΔΔCT method and normalized relative to the expression of glyceraldehyde-3-phosphate dehydrogenase using QuantStudio 6 and 7 Flex Real-Time PCR System Software in a QuantStudio™ 7 Flex System.\n\nAt 6 am, mice were fasted for 4 hr. Glucose levels were measured and blood samples were taken from the tail vein before oral glucose tolerance test (OGTT) was initiated. GTT was performed by oral administration of a bolus glucose (2g/kg body weight). Glucose levels were measured at 20, 40 and 60 min after glucose administration by using AlphaTrak blood glucose meter (Abbott, Chicago, IL).\n\nBlood samples were collected from tail vein when mice were at 11 weeks old, 15 weeks old (on HFD for 4 weeks) and 26 weeks old (on HFD for 15 weeks) Terminal blood was collected by cardiac puncture. Blood samples were centrifuged at 10000 rpm. Serum insulin levels were determined by using Insulin (mouse) ultra-sensitive EIA kit 80-INSMSU-E10 or mouse high range insulin ELISA 80-INSMSH-E01 (ALPCO Diagnostics, Salem, NH).\n\n\nResults and discussion\n\nWe isolated RNA from spleen, liver, perirenal fat (WAT) and brown fat (BAT) from Deltagen Gpr21 KO mice and their wildtype littermate control mice. Microarray results identified that Rabgap1 was the only gene that was changed in all the tissues analyzed. Rabgap1 mRNA levels were increased by 1–4 fold when using two independent Rabgap1 probes (Figures 1A, B, Table 1) located upstream of Gpr21 gene and were down regulated by 5–15 fold when using one Rabgap1 probe located downstream of Gpr21 gene. This result indicated that the genetic modifications in the original KO line modified Rabgap1 mRNA expression levels (Figure 1B). Strbp mRNA expression levels were not changed when using multiple probes that were located upstream and downstream of the Strbp gene (Figure 1A).\n\n(A) Mouse Gpr21 is located on Chromosome 2 within the intron of Rabgap1 gene between exon 13 and 14 on the positive strand according to UCSC GRCm38/mm10 assembly. Strpb gene is on the opposite strand in the same region. The blue arrow represents the positive strand while the green one the negative strand. The bars under the genes represent microarray probe sets from Affymetrix mouse array HT MG-430PM platform. There is no probe set covering Gpr21 gene. The closest probe set 1421125_PM is located at 2,866 bases upstream of Gpr21. (B) The level of Rabgap1 transcript was shown as normalized expression intensity. RNA was prepared from BAT, liver, spleen and WAT of Deltagen Gpr21 KO mice and their WT littermate controls. Probe 1443535_PM, 1460486_PM and 1424188_PM allow detection of Rabgap1 mRNA expression levels.\n\nA new line of Gpr21 KO mice (Gpr21 TAL 29bp) was created by deleting a 29 bp within the coding exon of Gpr21 gene. Using the TALENS technology, a 29 bp very close to the ATG codon was deleted, thus causing an out of frame mutation and early termination of Gpr21 gene (Figure 2A). Homozygous Gpr21 KO mice genotype was confirmed by PCR using primers located upstream and downstream of the 29bp deletion from genomic DNA of several tissues (Figure 2B). As predicted, a 100 bp band was identified for the wildtype mice and a 71 bp band for the Gpr21 TAL 29 bp homozygous KO mice (Figure 2C). The PCR fragment was sequenced and a 29 bp deletion was confirmed. From RNA isolated out of BAT and liver, no detectable Gpr21 mRNA levels were identified in Gpr21 TAL 29 bp KO mice using qPCR and Gpr21 probes that are located around the 29 bp deletion region (Figure 2D). Similar mRNA levels were detected using primer/probe that were located downstream of 29 bp deletion in exon 2 (data not shown). The result confirmed that Gpr21 transcripts in Gpr21 TAL 29bp KO mice had the 29 bp deletion.\n\n(A). Sequence and location of the 29 bp deletion in Gpr21 TAL 29bp KO mice. (B). sequence and location of genotyping primers. (C). Genotyping of Gpr21 TAL 29 bp KO mice. Genomic DNA was generated from liver and BAT. PCR with genotyping primers amplified a 100 bp fragment from the genome of WT littermate mice and a 71 bp fragment from homozygous Gpr21 TAL 29 bp KO mice. C: commercial mouse genomic DNA. L: 20 bp DNA ladder. (D). No wildtype Gpr21 transcript were detected. qPCR analysis of Gpr21 gene in liver and BAT using primer/probe set that is located in the 29 bp region and only detect wildtype Gpr21 transcript.\n\nRabgap1 mRNA expression levels were assessed using 2 Taqman probes (Table 1) that amplified different regions of Rabgap1 in liver and BAT of KO and their wildtype littermate mice. One primer/probe set spanned Rabgap1 exon 3 and 4 (Table 1), which is located upstream of the Gpr21 gene. Another primer/probe set spanned Rabgap1 exon 17 and 18, which is located downstream of the Gpr21 gene (Table 1). Rabgap1 mRNA expression levels in Gpr21 TAL 29 bp KO mice were not changed compared with their wildtype littermate mice with both primer/probe sets (Figure 3A). Liver and BAT Strbp mRNA expression levels were also not changed between Gpr21 TAL 29bp KO mice and their wildtype littermate mice (Figure 3B).\n\n(A) Rabgap1 mRNA expression levels were assessed using 2 Taqman probes in liver (top left panel) and BAT (top right panel) of GPR21 TAL 29 bp KO and their wildtype littermate mice. (B) Liver (bottom left panel) and BAT (bottom right panel) strbp mRNA expression levels were assessed in wildtype and Gpr21 TAL 29 bp KO mice.\n\nThe body weight, OGTT and insulin levels of Gpr21 TAL 29 bp KO mice fed a normal chow were not different from the ones of their wildtype littermates (Figures 4A–C). Mice were then fed with a 45% high fat diet to induce obesity and insulin resistance. After 4 weeks and 15 weeks of high-fat feeding, Gpr21 TAL 29 bp KO mice gained similar body weight to that of their wildtype littermates, showed no difference on glucose tolerance and fasting blood glucose and insulin levels were not different from their wildtype littermates (Figures 4D–I), respectively.\n\nAt 11 weeks of age, body weight (Figure 4A), OGTT (Figure 4B) and insulin levels (Figure 4C) were measured in wildtype (open bar and open circle) and Gpr21 TAL 29bp KO Mice (filled bar and open triangle) fed a normal chow diet. At 15 weeks of age and at 26 weeks of age body weight (Figures 4D & 4G), OGTT (Figures 4E & 4H) and insulin levels (Figures 4F & 4I) were measured in wildtype (open bar and open circle) and Gpr21 TAL 29bp KO Mice (filled bar and open triangle) fed a high fat diet for 4 weeks and 15 weeks, respectively.\n\nThe results of Osborn and Gardner suggest that GPR21 may play an important role in regulating body weight and glucose metabolism. However, in our attempts presented here to replicate their findings we didn’t see the same effect. We would therefore like to encourage an open discussion in the wider community to further elucidate the potential effectiveness of pharmacologically inhibiting GPR21.\n\n\nData availability\n\nOpen Science Framework: Dataset: GPR21 KO mice demonstrate no resistance to high fat diet induced obesity or improved glucose tolerance, doi: 10.17605/OSF.IO/SQB2X (Wang et al., 2016).",
"appendix": "Author contributions\n\n\n\nJW: study design, performed some experiments, wrote the manuscript; ZP: study design, performed some experiments; HB: study design; DC: generated TALENS KO mice, edited manuscript; CG: performed some experiments; MV: study design, wrote and edited the manuscript.\n\n\nCompeting interests\n\n\n\nAll authors were working at Amgen at the time the studies were performed.\n\n\nGrant information\n\nThis research was funded by Amgen Inc.\n\n\nReferences\n\nBenjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc. 1995; 57(1): 289–300. Reference Source\n\nBresnick JN, Skynner HA, Chapman KL, et al.: Identification of signal transduction pathways used by orphan g protein-coupled receptors. Assay Drug Dev Technol. 2003; 1(2): 239–249. PubMed Abstract | Publisher Full Text\n\nCuif MH, Possmayer F, Zander H, et al.: Characterization of GAPCenA, a GTPase activating protein for Rab6, part of which associates with the centrosome. EMBO J. 1999; 18(7): 1772–1782. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrasa MA, Koessmeier KT, Ahmadian MR, et al.: Illuminating the functional and structural repertoire of human TBC/RABGAPs. Nat Rev Mol Cell Biol. 2012; 13(2): 67–73. PubMed Abstract | Publisher Full Text\n\nGardner J, Wu S, Ling L, et al.: G-protein-coupled receptor GPR21 knockout mice display improved glucose tolerance and increased insulin response. Biochem Biophys Res Commun. 2012; 418(1): 1–5. PubMed Abstract | Publisher Full Text\n\nMiserey-Lenkei S, Couëdel-Courteille A, Del Nery E, et al.: A role for the Rab6A' GTPase in the inactivation of the Mad2-spindle checkpoint. EMBO J. 2006; 25(2): 278–289. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO'Dowd BF, Nguyen T, Jung BP, et al.: Cloning and chromosomal mapping of four putative novel human G-protein-coupled receptor genes. Gene. 1997; 187(1): 75–81. PubMed Abstract | Publisher Full Text\n\nOsborn O, Oh DY, McNelis J, et al.: G protein-coupled receptor 21 deletion improves insulin sensitivity in diet-induced obese mice. J Clin Invest. 2012; 122(7): 2444–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPires-daSilva A, Nayernia K, Engel W, et al.: Mice deficient for spermatid perinuclear RNA-binding protein show neurologic, spermatogenic, and sperm morphological abnormalities. Dev Biol. 2001; 233(2): 319–28. PubMed Abstract | Publisher Full Text\n\nWang J, Pan Z, Baribault H, et al.: Dataset: GPR21 KO mice demonstrate no resistance to high fat diet induced obesity or improved glucose tolerance. Open Science Framework. 2016. Data Source\n\nXiao SH, Reagan JD, Lee PH, et al.: High throughput screening for orphan and liganded GPCRs. Comb Chem High Throughput Screen. 2008; 11(3): 195–215. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "12620",
"date": "07 Mar 2016",
"name": "Mary Pelleymounter",
"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\nDr. Wang and colleagues have provided intriguing evidence to suggest that the metabolic phenotype of the Deltagen GPR21 KO mouse may not be completely due to deletion of the GPR21 gene. Rather, these authors suggest that the metabolic phenotype of the Deltagen GPR21 KO mouse could have been a function of the location of the 5.3 kb lacZ/Neo cassette within exon 1, which could have altered the expression and function of Rabgap1 and Strbp. Indeed, the authors did show that Rabgap1 was altered in brown and white adipose tissue, liver and spleen of the Deltagen GPR21 KO mice. In order to understand whether the metabolic phenotype of the Deltagen GPR21 KO mice was truly due to deletion of GPR21 or an artifact of the neo cassette location in the Deltagen mice, this group used a different strategy to delete GPR21 which did not involve Rabgap1 or Strbp. The KO mice generated as a result of this alternate GPR21 deletion strategy (GPR21 TAL29bp KO) did not demonstrate the high fat diet resistance phenotype observed in the Deltagen GPR21 KO mice. In addition, while there were no differences in Rabgap1 mRNA or strbp mRNA in the liver or brown adipose tissue of the GPR21 TAL 29bp KO mice, there was clear deletion of GPR21 in the same tissues of these mice.Although this information is quite important for the research area of obesity and diabetes therapeutics in general, there are some aspects of the experimental design and manuscript narrative that could be strengthened in order to provide more confidence in the findings.It is unclear why the authors chose to focus on liver and brown adipose tissue expression levels of Rabgap1 and GPR21. Tissue expression of GPR21 was much more prominent in brain and spleen in both the Gardner et al., and Osborn, et al., papers which described the high fat diet resistance phenotype in the Deltagen GPR21 KO mice. It seems that there would be even more confidence in the findings of the current manuscript if GPR21 was as dramatically knocked out in brain and spleen by the GPR21 TAL29bp deletion strategy as it was in liver and BAT. The argument that the improved metabolic phenotype in the Deltagen GPR21 KO mice was an artifact of neo cassette location would have been more convincing if the current authors had compared the Deltagen GPR21 KO mice in a “head to head” fashion with the GPR21 TAL29bp KO mice when they evaluated resistance to diet-induced impairments in glucose tolerance. This would have shown that the authors were able to reproduce the metabolic improvements previously observed in the high fat diet-fed Deltagen mice even though the GPR21 TAL29bpKO mice did not show such an improvement under the same experimental conditions. Since it is important to assure that the experimental conditions utilized to assess metabolic phenotype in the current study were similar to those utilized for the Deltagen GPR21 KO mice in other labs, it would have been helpful if the authors had provided more detail about their methodology in the current manuscript. For example, the following points are not clear from the narrative of the manuscript:a. What were the housing conditions of the animals (individual vs group-housed?, ambient temperature?b. Were the mice all one gender? (the Gardner paper used a mix of male and female, which could have influenced body weight, activity level, etc)c. What were the Ns for the OGTT, Insulin and BW data and how were they derived (basis for power analysis)?d. What statistical analysis strategy was used to evaluate the data?e. Was a baseline insulin level obtained under the conditions used for the OGTT?f. Were subsets of animals sacrificed after the respective OGTT and insulin measurements at each time point (11, 15 and 26 weeks)? If so, how was that taken into account in the statistical analysis for data collected over the entire time period (i.e., body weight)? In the “Next Steps” section, the authors suggest that the follow up to their data will be the responsibility of those that read their article. However, their data only opens up the possibility that the metabolic phenotype of the Deltagen GPR21 KO mice is an artifact of neocassette location. In this reviewer’s opinion, the authors still have quite a bit of work to do in order to provide definitive evidence that the Deltagen GPR21 KO mouse phenotype is not correct. It would also be informative to hear the author’s view on the potential role of Rabgap1 in insulin sensitivity and resistance.",
"responses": []
},
{
"id": "12759",
"date": "07 Mar 2016",
"name": "Richard Neubig",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title is appropriate. The design, methods, and analysis are generally complete and appropriate with the following exceptions:The methods to obtain data from the Deltagen GPR21 mutant mice are not sufficiently described. The source of the mice, genetic, background, and backcross history are not included in the methods. Since genetic background can profoundly change biological phenotypes, this information is essential to fully understand the differences between the Deltagen GPR21 KO and the new TALEN GPR21 KO mice described here. The full sequence of the PCR product across the 29 bp deletion should be shown along with an alignment with the Gpr21 gene sequence to ensure that the deletion is indeed within the Gpr21 gene. To definitively establish that the knockout does disrupt Gpr21, it would be helpful to additionally demonstrate reduced expression of the Gpr21 protein. This could be done by antibody detection methods, mass spec, or other functional assessments of Gpr21 activity. Minor correction suggested:Page 3 – “Strbp … probes that were located upstream and downstream of the Strbp gene … “ should probably read “downstream of the Gpr21 gene …”. The conclusions require modification and they should also be expanded to reflect broader implications of this study.The description of results in the “Next steps” paragraph should be amended. It is important in our discussions of “replication” that scientists be precise. The term replication should be used when the precise methods and reagents were used for the follow-on studies. This work does not “replicate” the original study but attempts to “confirm” or “extend” it using a different approach. Clearly robust scientific results must not only be replicable but also must be able to be confirmed or extended to have real value in advancing our understanding of biological results or drug action. I suggest the following. Replace “replicate their findings” with “confirm their findings using a Gpr21 KO mouse generated with a different technology”. The limitation that the Gpr21 protein levels were not assessed should be included in the discussion unless data are shown to address that limitation. It would also be valuable to note a more general conclusion from this study. In any work using genetically manipulated animal models, it is critical to demonstrate that the targeted manipulation is behind the biological differences being explored. While this should be obvious and generally assessed, this study illustrates one of the numerous ways in which scientists may be misled – changes in expression or function of other genes near the targeted gene. An analysis of the expression or function of nearby genes may be a general recommendation that could be made for all KO studies.",
"responses": []
},
{
"id": "12685",
"date": "24 Mar 2016",
"name": "Michelle Kimple",
"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 by Wang and colleagues addresses the resistance of GPR21 knockout mice to insulin resistance and high-fat diet induced obesity and metabolic dysregulation. Two previous publications using a different GPR21 knockout mouse model generated with ES cells from Deltagen have been previously published: Osborn et al. (2012) JCI 122: 2444-2453; and Gardner et al. (2012) BBRC 418: 1-5. Osborn et al. demonstrated that, at a baseline of 8 weeks, GPR21 KO mice were significantly lighter than their wild-type controls despite similar food intake, and after 11 weeks on normal chow had significantly improved insulin sensitivity and glucose tolerance. In addition, GPR21 KO mice fed a 60 kcal% HFD for 12 weeks showed no differences in % weight gained or adiposity on the diet, but maintained a significantly improved glucose tolerance and insulin sensitivity. Gardner et al. demonstrated that, at baseline of 120 days, GPR21 KO mice were lighter, again despite similar food intake, and after 12 weeks on a 45 kcal% diet, gained significantly less weight and adiposity and maintained better insulin sensitivity and glucose tolerance than the wild-type controls. In contrast, Wang and colleagues find no differences in body weight, adiposity, or glucose and insulin tolerance between their GPR21 knockout mice (generated using TALEN technology) and wild-type controls; an important finding they suggest is due to unintentional changes in RabGAP1 gene expression in the original Deltagen GPR21 knockout mice.General Comments:1) The aim of the current work, as stated in the last paragraph of the introduction, was to show that the effects observed in Osborn and Gardner were not solely due to loss of GPR21. I believe the authors have essentially demonstrated this, and the title of the article is appropriate and reflective of this. Yet, much of the abstract, introduction and results sections focus on RabGAP1 expression. More confirmatory experiments seem necessary to implicate changes in RabGAP1 in the Deltagen GPR21 knockout phenotype (see specific comment 2). 2) As a pure replicative paper, the authors did not directly replicate either of the original HFD studies performed in Osborn or Gardner. The 45 kcal% HFD protocol was closer to that described in Gardner, yet the mice were started on the HFD 6 weeks earlier (presumably at 11 weeks based on figure legends instead of ~17 weeks in Gardner) and continued on the diet 3 weeks longer. The statement in the \"Next Steps\" should be modified as such. Specific Comments:1) In the Materials and Methods section, the description of the high-fat diet experiments are not detailed enough. This is important as the high fat diet experiments in the original referenced publications are actually quite different from each other. Furthermore, this would allow other investigators to more easily replicate the diet study.2) The authors propose that changes in RabGAP1 expression might be responsible for the phenotypes observed in Gardner and Osborn. This conclusion would be stronger with qPCR analysis of the original Deltagen mouse tissues (such as in Figure 3, which was performed for the GPR21 TALEN KO mice) instead of reliance on microarray probe data. In such a case, a direct comparison of RabGAP1 expression could be made between the Deltagen and TALEN GPR21 knockout mice.3) Hypothalamic GPR21 expression was proposed as responsible for phenotype of mice on normal chow observed in Osborn et al, and GPR21 is highly expressed in the hypothalamus. I would recommend adding this tissue to the panel analyzed by qPCR in Figure 3.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-136
|
https://f1000research.com/articles/4-1461/v1
|
16 Dec 15
|
{
"type": "Software Tool Article",
"title": "Semi-automated Modular Program Constructor for physiological modeling: Building cell and organ models",
"authors": [
"Bartholomew Jardine",
"Gary M. Raymond",
"James B. Bassingthwaighte",
"Gary M. Raymond",
"James B. Bassingthwaighte"
],
"abstract": "The Modular Program Constructor (MPC) is an open-source Java based utility, built upon JSim's Mathematical Modeling Language (MML) (http://www.physiome.org/jsim/) that uses directives embedded in model code to construct larger, more complicated models quickly and with less error than manually combining models. A major obstacle in writing complex programs for modeling physiological processes is the large amount of time it takes to code the myriad processes taking place simultaneously in cells, tissues, and organs. MPC replaces this task by code-generating algorithms that take the code from several different modules and produce model code for a new JSim model. This is particularly useful during multi-scale model development where many variants are to be configured and tested against data. MPC is implemented in Java and requires JSim to use its output. MPC source code and documentation are available at http://www.physiome.org/software/MPC/.",
"keywords": [
"multi-scale modeling",
"JSim",
"systems biology",
"physiological modeling",
"reproducibility",
"uncertainty quantification"
],
"content": "Introduction\n\nMany attempts have been made to provide modular programming systems for physiological applications (Erson et al., 2012; Krause et al., 2010; Mirschel et al., 2009; Smith et al., 2009). We describe our system as a semi-automated modular programming construction. It is simple and not conceptually novel, but is easy to learn and use. For developing a series of models of increasing complexity, Modular Program Constructor (MPC) can serve well as the basis of the modeling code. The perspective is to take a modular approach; this means that for multi-scale modeling one builds from simple elements initially and then uses multi-modular constructs as modules in higher level systems.\n\nIn MPC, a module can be any set of variable declarations, parameter declarations and mathematical equations that represent a process. This broad definition of a module has a broad variety of applications: from a simple first order enzyme reaction, to a complete model of coronary blood flow through heart muscle, which can then be incorporated into a yet larger systemic model.\n\nBased on ModelBuilder, which used FORTRAN to parse code and define directives (Raymond, 2008), we designed the current version in Java and renamed it MPC. It is now refined, simplified, robust, and used to develop several new applications (Raymond et al., 2011; Raymond et al., 2012). The models include time-dependent two-dimensional models in both Cartesian and cylindrical coordinates (Raymond et al., 2011; Raymond et al., 2012), as well as whole organ models with heterogeneous flow re-implementing earlier complex whole organ models of substrate metabolism (Bassingthwaighte et al., 1989).\n\n\nMethods\n\nMPC is a pre-compiler written in Java. It reads a text input file, parses the file for directives, and generates a text output file based on those directives. MPC is built upon the Mathematical Modeling Language (MML) of JSim (http://www.physiome.org/jsim/) (Butterworth et al., 2014). It has been designed to work with JSim's MML and currently requires JSim to run the model output file that MPC produces. Through JSim, the final constructed model can be exported into Systems Biology Markup Language (SBML, http://sbml.org/Main_Page), CellML (https://www.cellml.org/), and downloaded from these sources to other simulation platforms (Smith et al., 2014). MPC currently is executed as command line utility and requires the Java runtime environment (https://java.com/).\n\nMPC has three components:\n\n1. MML, the mathematical modeling language of JSim, is a declarative language designed for solving all the equations simultaneously; it is not procedural. MML is used for declaring parameters and variables, for defining algebraic equations, ordinary differential equations, and partial differential equations with their associated constraints, and initial and boundary conditions.\n\n2. Modules, are sets of MML code libraries which are variable declarations, parameter declarations, or mathematical equations for a particular process, for example, flow along a capillary, diffusion within a region, a chemical reaction, transport across a membrane, or even a whole organ. These are archived, forming libraries of operational code. This allows the user to generate multi-scale models with different sub-models to use in testing a hypothesis against data, i.e. validity testing. For example, there have been a variety of models developed to describe the transmembrane sodium pump, NaKATPase which uses ATP to pump sodium out of, and potassium into, the cell. All of these models have the same essential external influences: the Na and K ion concentrations and the transmembrane electrical potential. Having a library of the MML code for the variant modules allows one to insert one's choice quickly into the template for the cell model. Changing combination(s) rapidly to match solutions with experimental results is invaluable for the early phases of developing alternative hypotheses.\n\n3. Directives, the third component, comprises the set of instructions used by the MPC program to select processes and gather the code from existing modules, renaming parameters and variables to reflect the new purposes for which they will function, and automatically combining the mathematical structures into new structures. The directives control the identification, fetching and relabeling of variables and parameters, and the assembly and recombination of code into new equations. All MPC directives start with '//%'.\n\nThe MPC input file guides the construction of a model made of previously existing modules. It combines MML with “directives” embedded as comments. It uses code from other JSim model files that have been annotated so that they can be read by MPC, yet without interfering with their operability. MPC may also combine models with other models or with modules of preconstructed code from libraries. These modules are specified within a library with the START and END directive. A library with a few elementary operators from which we will build a model in out next step is illustrated below:\n\nCodeLibrary.mod:\n\n\n\nIn JSim's MML, the colon signifies the derivative: C:t means dC/dt. Within MPC we can write MML code directly or import code from operational JSim models that have been annotated to identify components. An example is a three species (A, B, C), two compartment model with two reactions in compartment two (Figure 1) with species concentrations described by ordinary differential equations (ODE). Species A enters, with flow F, a compartment with volume V1 and passive exchange between a second compartment with volume V2, where A reacts at rate GA2B to form B and B reacts with C at rate GB2C, a Michaelis-Menten reaction.\n\nAin is A entering compartment 1 with flow F (No flow in for species B, C). Passive exchange between compartments for all three species and reactions only occur in compartment 2.\n\nThe MPC file defines the domain, parameters, variables, and initial conditions first. Using directives listed in ‘Example.mpc’, model code is extracted from the file ‘CodeLibrary.mod’ shown above. Values and variable names needing replacement throughout the final model are specified by the REPLACE directive along with the '%symbol%' placeholder. The use of the REPLACE, GET, COLLECT, INSERTSTART and INSERTEND directives are used in Example.mpc shown below:\n\nExample.mpc:\n\n\n\nThe GET directive warrants further explanation: it identifies a code library file and module name within the library to insert into the model, and changes old names (names of parameters and variables in the module) to new model names. From the example above, //%GET %CL% reactionCalc (\"A=A2\",\"B=B2\",\"V=V2\",\"G=Ga2b\") will get the module named 'reactionCalc' in file 'CodeLibrary.mod' and replace the variable names with the new model names (\"A=A2\", etc).\n\nThe MPC directives control the identification, fetching, relabeling of variables and parameters, and assembling and recombining code into new equations. The directives extract equations from files, changing the names of the module variables to application specific names and assemble the code into combined equations. The code resulting from these instructions provides a complete program (Example.mod), with no further intervention on the part of the user except to adjust parameters, solution time step length and set up graphics in JSim to display solutions, as shown in Figure 2.\n\nSpecies concentrations plotted as a function of time. Species A (red), B (green), C (blue). Compartment 1: solid line, Compartment 2: dashed line. External input for species A is Ain, the black solid line.\n\nMPC Output - Example.mod:\n\n\n\nThe process above is hardly worthwhile for small models but is highly efficient for larger models where flexibility in structure is desired. In the example above, converting the Ordinary Differential Equations (ODEs) to Partial Differential Equations (PDEs) requires a three line change. Addition of a new PDE e.g. for red blood cells in a capillary, takes four lines. For a five species, three region model, a three line change generates a 15 PDE model.\n\nThe small set of directives builds complex models from simple processes. MPC allows one to reliably reuse existing models in larger, multi-scale models. MPC encodes and preserves information about how a complex model is built from its modules allowing quick substitution of modules. The amount of actual code a user needs to write is reduced, especially for more complicated models. In MPC we have generated a full organ model with heterogeneity of flow, competitive transporters on the cell membranes, and reactions for multiple species (Bassingthwaighte et al., 2012) e.g. for adenosine processing in the heart. It is a 7-path, three region model that involves five species (adenosine, inosine, hypoxanthine, xanthine, and uric acid) in a sequential reaction chain. The model contains over 100 PDEs for convection, diffusion, and reactions.\n\n\nDiscussion\n\nThough a MPC-generated model is checked for syntax and unit balance through JSim, verification is required: analytical solutions can be written into the code to match specific limiting cases, but otherwise one depends on testing for mass, charge, or energy balances. Validation requires testing against data, independent of the construction method. These are key steps toward reproducibility and the VVUQ process. (VVUQ = verify, validate, uncertainty quantification; the latter defining predictive accuracy.) MPC as is, depends on semantic consistency throughout the libraries and models used. Automated systems using ontologies will help craft models (Gennari et al., 2011), but the great efficiency of MPC for construction begins to show when there are many modules in series/parallel arrangements as in biochemical networks or circulatory or airway mechanical modeling. UQ includes uncertainty in inputs and parameters, readily handled by JSim's Monte Carlo analysis, and in model structure. Structural uncertainty, a major challenge, defines a major role for MPC: inserting different choices from amongst similar but differently functioning modules, into a large, multi-modular model, and solving the system many times with the variant constituents illustrating uncertainty in the projected outcomes.\n\nA limited set of directives in MPC, our Modular Program Constructor, allows us to build complex models using small models for simple processes. MPC encodes and preserves information about how a complex model is built from its modules allowing the researcher to quickly substitute or update modules to validate a hypothesis. The amount of actual code a user needs to write is reduced, especially for more complicated models.\n\nFuture updates will improve collection and insertion of model code, better identify external module 'connections' for easier incorporation into larger models, and more intelligent reconciliation of similar code between modules. The long-term strategy is to integrate MPC within JSim allowing the user to take advantage of JSim's MML compiler and graphical user interface to quickly merge code with less user intervention.\n\n\nSoftware availability\n\nThe Java code for MPC, the examples presented here, some more detailed examples, and instructions are available at http://www.physiome.org/software/MPC/.\n\nhttps://github.com/F1000Research/MPC/releases/tag/v1.0\n\nhttp://dx.doi.org/10.5281/zenodo.34208\n\nMPC is released under a 3-clause ‘revised’ BSD license:\n\nCopyright (C) 1999–2015 University of Washington\n\nDeveloped by the National Simulation Resource\n\nDepartment of Bioengineering, Box 355061\n\nUniversity of Washington, Seattle, WA 98195-5061.\n\nDr. J. B. Bassingthwaighte, Director\n\nRedistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met:\n\n* Redistributions of source code must retain the above copyright notice, this list of conditions and the following disclaimer.\n\n* Redistributions in binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution.\n\n* Neither the name of the University of Washington nor the names of its contributors may be used to endorse or promote products derived from this software without specific prior written permission.\n\n\nData availability\n\nMPC generated models for review at www.physiome.org are:\n\n• Concentration profiles in capillary and tissue when exchange is diffusion-limited (http://www.physiome.org/jsim/models/webmodel/NSR/DiffusionLimitedProfiles/).\n\n• ODE model of actin polymerization and depolymerization with tracking of bound nucleotide (http://www.physiome.org/jsim/models/webmodel/NSR/ActinCycle1/).\n\n• Multiple tracer dilution estimates of D- and 2-deoxy-D-glucose uptake by the heart (http://www.physiome.org/jsim/models/webmodel/NSR/Kuikka1986BTEX30MP/).",
"appendix": "Author contributions\n\n\n\nGary Raymond developed MPC. Bart Jardine currently maintains MPC source code and James Bassingthwaighte provides guidance and requirements for MPC development. All authors contributed to the design and organization of the paper and its writing and editing. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declared no competing interests.\n\n\nGrant information\n\nResearch has been supported by NIH grants HL088516 (J.B. Bassingthwaighte) and HL073598 (J.B. Bassingthwaighte), BE08417 (J.B. Bassingthwaighte), the Virtual Physiological Rat program GM094503 (PI: D.A. Beard), and the Cardiac Energy Grid HL199122 (PI: J.B. Bassingthwaighte). Grants supported whole group.\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\nBassingthwaighte JB, Wang CY, Chan IS: Blood-tissue exchange via transport and transformation by capillary endothelial cells. Circ Res. 1989; 65(4): 997–1020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBassingthwaighte JB, Raymond GM, Chan JI: Tracer washout from an organ is predicted from the tracer center of mass. FASEB J. 2012; 26: 905.16. Reference Source\n\nButterworth E, Jardine BE, Raymond GM, et al.: JSim, an open-source modeling system for data analysis [version 3; referees: 2 approved]. F1000Res. 2014; 2: 288. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErson EZ, Cavuşoğlu MC: Design of a framework for modeling, integration and simulation of physiological models. Comput Methods Programs Biomed. 2012; 107(3): 524–37. PubMed Abstract | Publisher Full Text\n\nGennari J, Neal ML, Galdzicki M, et al.: Multiple ontologies in action: Composite annotations for biosimulation models. J Biomed Informatics. 2011; 44(1): 146–154. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKrause F, Uhlendorf J, Lubitz T, et al.: Annotation and merging of SBML models with semanticSBML. Bioinformatics. 2010; 26(3): 421–422. PubMed Abstract | Publisher Full Text\n\nMirschel S, Steinmetz K, Rempel M, et al.: PROMOT: modular modeling for systems biology. Bioinformatics. 2009; 25(5): 687–689. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRaymond GM: Reusable modular code for multi-scale physiological systems modeling. Oral presentation 3rd MEI International symposium. 2008. Reference Source\n\nRaymond GM, Bassingthwaighte JB: Automating modular model construction using JSim. FASEB J. 2011; 25: 863.9. Reference Source\n\nRaymond GM, Bassingthwaighte JB: JSim models of two-dimensional concentrations in capillary-tissue systems relating center-of-mass of retained tracer to washout kinetics. FASEB J. 2012; 26: 905.17. Reference Source\n\nSmith LP, Bergmann FT, Chandran D, et al.: Antimony: a modular model definition language. Bioinformatics. 2009; 25(18): 2452–2454. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith LP, Butterworth E, Bassingthwaighte JB, et al.: SBML and CellML translation in Antimony and JSim. Bioinformatics. 2014; 30(7): 903–907. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11594",
"date": "05 Jan 2016",
"name": "Dagmar Waltemath",
"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 at hand describes MPC, a tool that supports modelers in constructing complex models from smaller ones. MPC also keeps information about the single modules, making their exchange and further coupling even easier.The manuscript provides several examples (on code and abstract level) of how to use MPC, but it does not give details on how the algorithm itself works.My suggestions for improvements are mainly on the terminology used throughout the manuscript, and on the discussion of related work.Unifying terms: In the abstract alone you speak about programs, utilities, code; about models, processes, model code and modules. Maybe you could - not only in the abstract but throughout the manuscript - unify your wording a little bit more to make the text more comprehensive. Related work: I missed a discussion of related systems, e.g. the model merge tool for SBML, semanticSBML, or the semantic-based system (there was a new publication just recently1). While you mention them in the beginning of your introduction, I did not see a discussion of these systems, and how they differ from your approach. I, as a reader, would be interested to know which system is best to use when. Furthermore, I have the following smaller comments: Page 2, Introduction: \"The models include time-dependent...\" -- Here it was not clear to me what you mean by \"models\". Page 2, MPC implementation: \"Through JSim, the final constructed model....\" -- I understand here, that you can upload your constructed models from JSim into an open model repository, and then directly download them into other simulation platforms. I am not sure that it is as easy as this, particularly for BioModels there will be a curation process in between, and there is thus no immediate reuse. The way the sentence is written now, a reader may assume that models can directly and immediately be exchanged through these resources, which is in my opinion misleading. Page 2, MPC implementation: \"These are archived, forming libraries of operational code\" -- I would be interested to know how you archive the modules, where, and how/if/to what degree they are accessible/reusable? Figure 1: I would like to suggest using an SBGN-compliant notation for the toy model. Summary: \"MPC encodes and preserves...\" -- This is an important information for the users, and I would like to suggest to add this information to the abstract.",
"responses": [
{
"c_id": "1907",
"date": "07 Apr 2016",
"name": "Bartholomew Jardine",
"role": "Author Response",
"response": "Our responses to Referee Dagmar Waltemath's review:My suggestions for improvements are mainly on the terminology used throughout the manuscript, and on the discussion of related work. Unifying terms: In the abstract alone you speak about programs, utilities, code; about models, processes, model code and modules. Maybe you could - not only in the abstract but throughout the manuscript - unify your wording a little bit more to make the text more comprehensive.Author Response: Yes, we updated the abstract and paper as a whole to try to use consistent and unifying wording when discussing model code, processes, modules, etc. These changes are most notable in the abstract and introduction. Related work: I missed a discussion of related systems, e.g. the model merge tool for SBML, semanticSBML, or the semantic-based system (there was a new publication just recently1). While you mention them in the beginning of your introduction, I did not see a discussion of these systems, and how they differ from your approach. I, as a reader, would be interested to know which system is best to use when.Author Response: Added a paragraph in the Introduction that briefly discusses other tools in relation to MPC:\"Modular model creation and construction rely, to varying degrees, on meta-data to assist in reusing and merging previous models into a new one. Antimony (Smith 2009) is the simplest approach. It requires the user to be familiar with the model and just specify that you want to import it into the new model. It relies on the user to resolve discrepancies between models. SemanticSBML(Krause 2010), SemGen (Genari 2011, Neal 2015), and Phy-Sim (Erson 2012) make use of standard semantic and ontological descriptions of a biological model to allow large models to be broken down easily, without much user guidance, into biologically meaningful components linked to their mathematical description. Semantic and ontological metadata assists the construction of new models by providing suggested connections or relationships between models. This approach requires the user to invest time in complete annotation of models with standardized meta-data. The payoff is models that can be constructed and merged together using biological rather than mathematical terms. ProMot (Mirschel 2009) enforces an object-oriented approach to modeling (defining external interfaces for each object) and attempts to use network theory to describe biological systems through specifying elements and coupling elements (Mirschel 2009). MPC relies on the user to modularize a model using directives to specify them. MPC then requires the user to specify how the new model makes use of the modules. MPC only imposes unit balance constraints, indirectly, through the JSim MML compiler (Butterworth 2014).\"Furthermore, I have the following smaller comments:Page 2, Introduction: \"The models include time-dependent...\" -- Here it was not clear to me what you mean by \"models\".Author Response: Clarified sentence to make it clearer (Page 2, Introduction, 4th paragraph):Some MPC built models include time-dependent two-dimensional spatial models in both Cartesian and cylindrical coordinates (Raymond et al., 2011; 2012), requiring PDEs, and whole organ models with heterogeneous flows, and substrate metabolism, including reconstructing Bassingthwaighte et al., 1989 blood-tissue exchange model. Page 2, MPC implementation: \"Through JSim, the final constructed model....\" -- I understand here, that you can upload your constructed models from JSim into an open model repository, and then directly download them into other simulation platforms. I am not sure that it is as easy as this, particularly for BioModels there will be a curation process in between, and there is thus no immediate reuse. The way the sentence is written now, a reader may assume that models can directly and immediately be exchanged through these resources, which is in my opinion misleading.Author Response: That sentence is confusing and not what we wanted to say. Changed to:Through JSim, the final constructed model can be exported into Systems Biology Markup Language (SBML, http://sbml.org/Main_Page) or CellML (https://www.cellml.org/), and imported to other SBML or CellML supported simulation platforms [Smith et al., 2014]. Page 2, MPC implementation: \"These are archived, forming libraries of operational code\" -- I would be interested to know how you archive the modules, where, and how/if/to what degree they are accessible/reusable?Author Response: Modules created and used by our team are currently available for download at physiome.org (http://physiome.org/software/MPC/) or search on term \"mpc\" (http://physiome.org/Models/modelDB/). At this time there are a very limited set of MPC modules available. Soon (May/June 2016) we will have individual MPC annotated modules accessible directly from our search page with all file dependencies listed and available for download as well as links to full JSim models that may use them. Contributions to our model repository are encouraged (Any modeling language accepted).Sentence inserted on page 2, in MPC implementation, paragraph 2, Modules: \"These are archived, forming libraries of operational module code that can be publicly distributed (some are available at http://www.physiome.org/software/MPC/).\" Figure 1: I would like to suggest using an SBGN-compliant notation for the toy model.Author Response: Thank you for the suggestion, for this particular figure we would like to keep it as is, but since we are currently modeling cardiac metabolism at the sub-cellular level we will be adopting SBGN notation where possible. Arrowheads in figure are made smaller. Summary: \"MPC encodes and preserves...\" -- This is an important information for the users, and I would like to suggest to add this information to the abstract.Author Response: Added this sentence to the abstract."
}
]
}
] | 1
|
https://f1000research.com/articles/4-1461
|
https://f1000research.com/articles/5-1397/v1
|
16 Jun 16
|
{
"type": "Research Article",
"title": "High prevalence of diabetes mellitus and impaired glucose tolerance in liver cancer patients: A hospital based study of 4610 patients with benign tumors or specific cancers",
"authors": [
"Chen Roujun",
"Yi Yanhua",
"Li Bixun",
"Chen Roujun",
"Yi Yanhua"
],
"abstract": "Objective: The prevalence of diabetes mellitus (DM), impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) were hypothesised to be different among different tumor patients. This study aimed to study the association between the prevalence of DM, IGT and IFG and liver cancer, colorectal cancer, breast cancer, cervical cancer, nasopharyngeal cancer and benign tumor. Methods: A hospital based retrospective study was conducted on 4610 patients admitted to the Internal Medical Department of the Affiliated Tumor Hospital of Guangxi Medical University, China. Logistic regression was used to examine the association between gender, age group, ethnicity , cancer types or benign tumors and prevalence of DM, IFG, IGT. Results: Among 4610 patients, there were 1000 liver cancer patients, 373 breast cancer patients, 415 nasopharyngeal cancer patients, 230 cervical cancer patients, 405 colorectal cancer patients, and 2187 benign tumor patients. The prevalence of DM and IGT in liver cancer patients was 14.7% and 22.1%, respectively. The prevalence of DM and IGT was 13.8% and 20%, respectively, in colorectal cancer patients, significantly higher than that of benign cancers. After adjusting for gender, age group, and ethnicity, the prevalence of DM and IGT in liver cancers patients was 1.29 times (CI :1.12-1.66) and 1.49 times (CI :1.20-1.86) higher than that of benign tumors, respectively. Conclusion: There was a high prevalence of DM and IGT in liver cancer patients.",
"keywords": [
"Prevalence",
"Diabetes mellitus",
"Impaired glucose tolerance",
"Liver cancer",
"Risk Factor"
],
"content": "Background\n\nHigh fat and carbohydrate diets, associated with dietary carcinogenesis, contribute to abnormal glucose tolerance and cancer1,2. Previous studies suggest that metabolic syndrome is associated with a modestly increased risk of second breast cancer events and breast cancer-specific mortality3. Blood glucose level in colorectal cancer patients has been shown to correlate significantly with local tumor malignancy4. Antidiabetic medication such as metformin treatment could significantly lower the risk of colorectal cancer in type 2 diabetes mellitus (T2DM) patients5. However, little is known on the correlation between prevalence of impaired glucose tolerance (IGT), diabetes mellitus (DM) and other types of cancers in comparison with benign tumors.\n\nAs an ethnic area, Guangxi Zhuang automonous region (abbreviated as Guangxi) located in southwest China, is well known for its high incidence of liver cancer and nasopharyngeal cancer6,7. Established risk factors including heavy alcohol consumption, chronic infection with the hepatitis B virus (HBV) or the hepatitis C virus (HCV), tobacco smoking, intake of aflatoxin-contaminated foods for the development of hepatocellular carcinoma (HCC) have been studied in Guangxi over the past several decades8. According to an American study, abnormal glucose tolerance was an independent predictor for cancer mortality9. Therefore, it is essential to investigate the prevalence of DM, IGT, and IFG (impaired fasting glucose) among patients with common types of cancer in such a high incidence area.\n\nThis study aimed to investigate the association between the prevalence of DM, IGT and IFG and liver cancer, colorectal cancer, breast cancer, cervical cancer, nasopharyngeal cancer and benign tumors.\n\n\nMethod\n\nThis hospital based study was approved by the Medical Ethics Committee of the Affiliated Tumor Hospital of Guangxi Medical University, China. The data were retrieved from the Internal Medical Department of the hospital. All personal identification was encrypted.\n\nPatients hospitalized in the Internal Medical Department between 2010 and 2012 with complete records of fasting plasma glucose (FPG) and 2-h postprandial glucose (2hPPG) upon hospitalization were included in this study. Clinical data were collected, including gender, age, ethnicity, DM history, diagnosis, pathological diagnosis, FPG, and 2hPPG. All diagnoses of cancers and benign tumors were based on CT scan results, endoscopic biopsy or surgical resection. Those who had serious cardiocelebral diseases, liver or kidney dysfunction and other conditions that may influence diabetic or prediabetic state were excluded from this study.\n\nThe glucose levels were included in the retrospective data. The 1999 World Health Organization (WHO) diagnostic criteria were used to diagnose DM, IFG and IGT10. Results of plasma glucose testing were categorized as follows. Normal glucose tolerance (NGT): FPG <6.1 mmol/L, and 2hPPG <7.8 mmol/L; Diabetes mellitus (DM): FPG ≥7 .0 mmol/L, 2hPPG ≥11.1 mmol/L, and with DM medical history. Impaired Fasting blood glucose (IFG): FPG ≥6.1 mmol/L and <7.0 mmol/L with the exclusion of cases with DM history or 2hPPG ≥11.1 mmol/L. Impaired glucose tolerance (IGT): FPG<7.0 mmol/L, and 2hPPG ≥7.8 mmol/L and <11.1 mmol/L with the exclusion of cases with DM history.\n\nDescriptive data were presented as frequencies and percentages, followed by a chi-square test or Fisher’s test as appropriate. Logistic regression was used to examine the association between gender, age group, ethnicity and cancer types or benign tumors and prevalence of DM, IFG, IGT. Data analyses were performed using the R language and environment (version 3.1.0)11 and the Epicalc package (version 3.1.1.2)12.\n\n\nResults\n\n1. Demographic description and prevalence of DM, IGT, and IFG among different cancers and benign tumors:\n\nA total of 4610 patients were included in this study. Figure 1 and Table 1 summarize the demographic characteristics of the patients, among whom 1000 were liver cancer patients, 373 breast cancer patients, 415 nasopharyngeal cancer patients, 230 cervical cancer patients, 405 colorectal cancer patients, and 2187 benign tumor patients. The patients in the 40–60 year age group comprised more than 50% of the total patients studied. Liver cancer, nasopharyngeal cancer, and colorectal cancer patients were predominantly male. Age and ethnic distribution among the studied groups were statistically different (P<0.001).\n\nData were given as n (%). * p-value<0.05; ** p-value<0.01; *** p-value < 0.001.\n\nThe prevalence of DM and IGT in liver cancer patients was 14.7% and 22.1%, respectively; 13.8% and 20% in colorectal cancer patients, which were significantly higher than that of benign cancers.\n\n2. Association between prevalence of DM and cancer types and benign tumors\n\nAs shown in Table 2, adjusted for gender, ethnicity, and age group, the prevalence of DM in liver cancers patients was 1.29 times higher than that of benign tumors; male patients were 1.49 times of higher than that of female patients. Using the 0–30 year as reference, the prevalence of DM increased with age. The peak risk ratio was 10.3 times higher in the 61–70 year age group than that of 0–30 year age group. The Han ethnic group was 1.38 times more likely to have DM in comparison with the Zhuang ethnic group.\n\n3. Association between prevalence of IGT and cancer types and benign tumors\n\nAs shown in Table 3, adjusted for gender, ethnicity, and age group, the prevalence of IGT in liver cancer patients was 1.49 times higher than that of benign tumors; male patients were 1.71 times higher than that of female patients. Using the 0–30 year as reference, there were an obvious age-dependent relationship between age groups and prevalence of IGT. There were no significant difference among the ethnic groups in terms of IGT prevalence (P=0.421).\n\n4. Association between prevalence of IFG and cancer types and benign tumors\n\nAs shown in Table 4, adjusted for gender, ethnicity, and age group, the prevalence of IFG in liver cancers patients was 0.55 times lower than that of benign tumors. Using the 0–30 year as reference, the prevalence of IFG increased with age. The Han ethnic group was 1.59 times higher in prevalence of IFG in comparison with the Zhuang ethnic group.\n\n\nDiscussion\n\nThis study showed that the prevalence of DM and IGT in liver cancer patients was 14.7% and 22.1%, respectively. After adjustment for gender, age group, and ethnicity, the prevalence of DM and IGT in liver cancer patients was 1.29 times (CI :1.12–1.66) and 1.49 times (CI :1.20–1.86) higher, respectively, than that of benign tumors. In comparison, the prevalence of DM and IGT in patients with breast cancer, cervical cancer, colorectal cancer, and nasopharyngeal cancer was not significantly different from that of benign tumors after adjusted for gender, age group, and ethnicity.\n\nA previous study suggested high blood glucose levels in colorectal cancer patients with larger tumor diameters and lower tumor differentiation4. Our study also found that the prevalence of DM and IGT was high in colorectal cancer patients in the preliminary analysis, although no statistical difference was found after adjustments for other factors. So far, there have been very few studies on the relationship between prevalence of DM and IGT and liver cancer. However, the relationship between obesity, overweight and higher rates of death due to cancer of esophagus, colon, rectum, liver, gallbladder, pancreas and kidney, non-Hodgkin’s lymphoma, and multiple myeloma has been well established by a large sample prospective study13. The reason why a high prevalence of DM and IGT is correlated with liver cancer may be explained as follows: As a regulator of energy storage and metabolism, insulin was produced and secreted by pancreatic β cells, which stimulated glucose uptake by adipose tissue and muscle, suppressing the release of glucose from the liver. Desensitization of tissues to insulin and insulin resistance led to a compensating increase in pancreatic insulin production called hyperinsulinemia, which may have possible direct oncogenic effects on proliferative and anti-apoptosis signaling in cancer cells.\n\nHigh prevalence of DM and IGT correlated with liver cancer had important implications both in prevention and treatment of liver cancer. Metformin, a commonly used drug for type 2 diabetes, led to a lower incidence and mortality of breast cancer14–18. In a similar way, the control of high blood glucose levels would possibly improve clinical outcomes of liver cancer patients.\n\nAs a hospital based study recently conducted in Guangxi with a high incidence of liver cancers, this study provided evidence for government and public health planners which may be useful in the prevention of liver cancer. Further prospective cohort studies should be conducted to learn the strength of association between DM, IGT and liver cancer; moreover, to learn how much the control of diabetic or pre-diabetic conditions contributes to the prevention and treatment of liver cancer.\n\n\nConclusion\n\nThis study showed a high prevalence of DM and IGT in liver cancer patients. The control of diabetic or pre-diabetic conditions may contribute to the prevention and treatment of liver cancer.\n\n\nData and software availability\n\nF1000Research: Dataset 1. Patient data, 10.5256/f1000research.8457.d12289419\n\nF1000Research: Dataset 2. Data analysis using R, 10.5256/f1000research.8457.d12289520",
"appendix": "Author contributions\n\n\n\nRC was principal investigator of the study, conceptualized the research, collected the data, performed data analysis, and drafted the manuscript. YY and BL conceived the study, assisted in development of data analysis, manuscript writing, and provided supervision and suggestions.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe research is supported by the Grant of Guangxi Science Research and Technology Development (No:1598012-8) to Bixun Li.\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\nSchoen RE, Tangen CM, Kuller LH, et al.: Increased blood glucose and insulin, body size, and incident colorectal cancer. J Natl Cancer Inst. 1999; 91(13): 1147–1154. PubMed Abstract | Publisher Full Text\n\nLu LJ, Wang RJ, Ran L, et al.: On the status and comparison of glucose intolerance in female breast cancer patients at initial diagnosis and during chemotherapy through an oral glucose tolerance test. PLoS One. 2014; 9(4): e93630. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCalip GS, Malone KE, Gralow JR, et al.: Metabolic syndrome and outcomes following early-stage breast cancer. Breast Cancer Res Treat. 2014; 148(2): 363–377. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCui G, Zhang T, Ren F, et al.: High Blood Glucose Levels Correlate with Tumor Malignancy in Colorectal Cancer Patients. Med Sci Monit. 2015; 21: 3825–3833. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang ZJ, Zheng ZJ, Kan H, et al.: Reduced risk of colorectal cancer with metformin therapy in patients with type 2 diabetes: a meta-analysis. Diabetes Care. 2011; 34(10): 2323–2328. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeng W, Long L, Li JL, et al.: Mortality of major cancers in Guangxi, China: sex, age and geographical differences from 1971 and 2005. Asian Pac J Cancer Prev. 2014; 15(4): 1567–1574. PubMed Abstract | Publisher Full Text\n\nZhang CY, Huang TR, Yu JH, et al.: Epidemiological analysis of primary liver cancer in the early 21st century in Guangxi province of China. Chin J Cancer. 2010; 29(5): 545–550. PubMed Abstract | Publisher Full Text\n\nYu MC, Yuan JM, Lu SC: Alcohol, cofactors and the genetics of hepatocellular carcinoma. J Gastroenterol Hepatol. 2008; 23(Suppl 1): S92–S97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaydah SH, Loria CM, Eberhardt MS, et al.: Abnormal glucose tolerance and the risk of cancer death in the United States. Am J Epidemiol. 2003; 157(12): 1092–1100. PubMed Abstract | Publisher Full Text\n\nShaw JE, de Courten M, Boyko EJ, et al.: Impact of new diagnostic criteria for diabetes on different populations. Diabetes Care. 1999; 22(5): 762–766. PubMed Abstract | Publisher Full Text\n\nR Core Team: R: A Language and Environment for Statistical Computing. 2015. Reference Source\n\nChongsuvivatwong V: Epicalc: Epidemiological calculator. R package version 2.15.1.0. 2012. Reference Source\n\nCalle EE, Rodriguez C, Walker-Thurmond K, et al.: Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. New Engl J Med. 2003; 348(17): 1625–1638. PubMed Abstract | Publisher Full Text\n\nBosco JL, Antonsen S, Sørensen HT, et al.: Metformin and incident breast cancer among diabetic women: a population-based case-control study in Denmark. Cancer Epidemiol Biomarkers Prev. 2011; 20(1): 101–111. PubMed Abstract | Publisher Full Text\n\nChoi Y, Kim TY, Oh DY, et al.: The impact of diabetes mellitus and metformin treatment on survival of patients with advanced pancreatic cancer undergoing chemotherapy. Cancer Res Treat. 2016; 48(1): 171–179. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeCensi A, Puntoni M, Goodwin P, et al.: Metformin and cancer risk in diabetic patients: a systematic review and meta-analysis. Cancer Prev Res (Phila). 2010; 3(11): 1451–1461. PubMed Abstract | Publisher Full Text\n\nEvans JM, Donnelly LA, Emslie-Smith AM, et al.: Metformin and reduced risk of cancer in diabetic patients. BMJ. 2005; 330(7503): 1304–1305. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoodwin PJ, Stambolic V, Lemieux J, et al.: Evaluation of metformin in early breast cancer: a modification of the traditional paradigm for clinical testing of anti-cancer agents. Breast Cancer Res Treat. 2011; 126(1): 215–220. PubMed Abstract | Publisher Full Text\n\nYi Y, Roujun C, Guijie H, et al.: Dataset 1 in: High prevalence of diabetes mellitus and impaired glucose tolerance in liver cancer patients: A hospital based study of 4610 patients with benign tumors or specific cancers. F1000Research. 2016. Data Source\n\nYi Y, Roujun C, Guijie H, et al.: Dataset 2 in: High prevalence of diabetes mellitus and impaired glucose tolerance in liver cancer patients: A hospital based study of 4610 patients with benign tumors or specific cancers. F1000Research. 2016. Data Source"
}
|
[
{
"id": "14413",
"date": "24 Jun 2016",
"name": "He Jianhui",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 “High prevalence of diabetes mellitus and impaired glucose tolerance in liver cancer patients: A hospital based study of 4610 patients with benign tumors or specific cancers” is very meaningful on providing evidence for government to prevent liver cancer in China. They found that the prevalence of DM and IGT in liver cancers patients was significantly higher than in benign tumors. Overall, I appreciate the study. The following is some comments and suggestions on the study topic.\nSome studies1-3 showed that DM is an independent risk factor of hepatocellular carcinoma, but some other studies4,5 provided an inconsistent conclusion: only among people who were infected with both HBV and HCV or alcoholic hepatitis can this increase the risk of hepatocellular carcinoma. Furthermore, a systematic review6 pointed out that among 23 case control or follow up studies, only less than 40% of studies indicated that DM is an independent risk factor of hepatocellular carcinoma, and also few studies can exclude the influence of food habits and obesity etc. The reason for this inconsistency is not clear, and needs further study. DM possibly is not an independent risk factor on hepatocellular carcinoma, but among people who were hepatitis virus infected or alcoholic with DM will increase the probability getting hepatocellular carcinoma.\nAccording to the above, I suggest that, if available, please add the information about some habits such as alcoholic beverage drinking history, some indices including BMI for example to exclude the influence of such factors which are both related to DM and hepatocellular carcinoma, and the status of virus hepatitis in the analysis. Moreover, a retrospective study cannot provide the causal relationship between DM and hepatocellular carcinoma, if we want to know the relationship clearly, a population based case control and prospective cohort study are needed.",
"responses": []
},
{
"id": "14411",
"date": "23 Aug 2016",
"name": "Nirun Intarut",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a retrospective study that was carried out in a hospital in China, involving 4610 participants. In summary, this study investigated the association between the DM, IGT and IFG and liver cancer, colorectal cancer, breast cancer, cervical cancer, nasopharyngeal cancer and benign tumors. The study shows the high prevalence of DM and IGT in liver cancer.\nMy suggestion if possible, is to include tobacco consumption or exposure to secondhand smoke into this study. Because there is a high prevalence of smoking and also high exposure to secondhand smoke in China 1-3, there could be evidence to show its association between DM and liver cancer 4-7.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1397
|
https://f1000research.com/articles/3-262/v2
|
13 Nov 14
|
{
"type": "Research Note",
"title": "DOCLASP - Docking ligands to target proteins using spatial and electrostatic congruence extracted from a known holoenzyme and applying simple geometrical transformations",
"authors": [
"Sandeep Chakraborty"
],
"abstract": "The ability to accurately and effectively predict the interaction between proteins and small drug-like compounds has long intrigued researchers for pedagogic, humanitarian and economic reasons. Protein docking methods (AutoDock, GOLD, DOCK, FlexX and Glide to name a few) rank a large number of possible conformations of protein-ligand complexes using fast algorithms. Previously, it has been shown that structural congruence leading to the same enzymatic function necessitates the congruence of electrostatic properties (CLASP). The current work presents a methodology for docking a ligand into a target protein, provided that there is at least one known holoenzyme with ligand bound - DOCLASP (Docking using CLASP). The contact points of the ligand in the holoenzyme defines a motif, which is used to query the target enzyme using CLASP. If there are no significant matches, the ligand cannot be docked in the protein. Otherwise, the holoenzyme and the target protein are superimposed based on congruent atoms. The same linear and rotational transformations are also applied to the ligand, thus creating a unified coordinate framework having the holoenzyme, the ligand and the target enzyme. This provides the docked ligand in the target enzyme. Previously, CLASP was used to predict and validate (in vivo) the inhibition of phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus by two dipeptidyl peptidase-IV (DPP4) inhibitors - vildagliptin and K-579. In the current work, vildagliptin was docked to the PI-PLC structure complexed with myo-inositol using DOCLASP. The docked ligand is free from steric clashes and interacts with the same side chain residues that bind myo-inositol, providing corroboration of the validity of the proposed methodology.",
"keywords": [
"protein",
"docking ligand",
"congruence"
],
"content": "Introduction\n\nThe ability to computationally predict protein-ligand interactions with accuracy is an invaluable asset, since it allows for large scale screening at minimal costs1,2. Consequently, computational methods that predict the favorable conformation of a protein-ligand complex have been the focus of intense research over the last few decades3. Protein docking methods are a subset of these methods, characterized by their ability to score a large number of possible conformations using fast algorithms. Among these, five programs - AutoDock4, GOLD5, DOCK6, FlexX7 and Glide8 - are the most cited, although it should be noted that ‘the number of citations of a given paper is no measure of quality of the corresponding protein-ligand docking software program’9. Typically, a protein-ligand docking program has two distinct phases - conformational sampling (or searching) and scoring10. Despite the significant progress in the field, there are several challenges arising from protein or ligand flexibility, entropic considerations or the presence of water molecules that need to be addressed11.\n\nPreviously, the conservation of spatial and electrostatic properties in cognate pairs of residues in the catalytic site of proteins with the same functionality has been used to develop a computational method (CLASP) for detecting binding and catalytic sites12–15. In the current work, this methodology has been extended by proposing a method for docking ligands into target proteins - DOCLASP (Docking using CLASP). DOCLASP takes as input a set of proteins with known structures which bind a particular ligand, and a target protein into which the ligand is to be docked. Each of these holo structures is used to define a motif consisting of the first four residues making non-hydrophobic interactions. These motifs are used to query the target protein, using an enhanced version of the search engine used by CLASP that uses precompiled databases16, and significant congruent matches are identified. These significant matches in the target protein are now superimposed to the binding residues (the motif) in the corresponding holoenzyme(s), thus creating a unified coordinate framework formed by the holoenzyme, the ligand and the target enzyme. This gives us the docked ligand to the target protein, which is outputted as a Pymol formatted file. In essence, the holoenzyme is replaced with the target enzyme if the contact points have a good spatial and electrostatic match in the target enzyme by aligning the congruent atoms. Thus, DOCLASP leverages the implicit search and scoring functions in CLASP to rank possible conformations.\n\nThe native activity of phosphoinositide-specific phospholipase C (PI-PLC) was previously shown to be inhibited by two dipeptidyl peptidase-IV (DPP4) inhibitors - vildagliptin (LAF-237) at micromolar concentrations, and K-579 at nanomolar concentrations using in vitro experiments based on CLASP analysis15. Since ‘comparing docking programs can be difficult’9, the DOCLASP methodology is validated by docking vildagliptin to the PI-PLC structure in complex with myo-inositol17. The docked ligand is free from steric clashes and interacts with the exact side chain residues that bind myo-inositol, providing corroboration of the validity of the proposed methodology. Thus, the current work presents a fast methodology for docking ligands into protein structures based on spatial and electrostatic congruence of known binding sites to putative binding targets.\n\n\nMaterials and methods\n\nDOCLASP uses the basic hypothesis of CLASP - the non-triviality of the spatial and electrostatic congruence in cognate pairs seen across different structures of the same catalytic function, which is extended to the related concept of ligand binding12. It takes as input a set of M proteins with known structures (Equation 1) which bind a particular ligand (Lig), and a target protein into which Lig is to be docked (Ptarget). Each of these M holo structures is used to define a motif consisting of N (=4) residues (Equation 2), taking the first four closest non-hydrophobic interactions into account (Algorithm 1).\n\n\n\n\n\nInput: Protein :\n\nInput: Ligand :\n\nInput: n: number of closest atoms to choose\n\nOutput: ϕmotif = {atom1 … atomn}\n\nbegin\n\n/* Output Motif */\n\nϕmotif = ∅ ;\n\n/* Accepted atom pairs - exclude hydrophobic interactions*/\n\nϕAcceptedAtomPair = [O-N, N-O, O-H, H-O, O-O, N-N, N-H, H-N, S-H, H-S] ;\n\nATOMSLig = atoms of all residues of Ligand ;\n\n/* Initial radius in Å */\n\nRadius = 2.5 ;\n\nforeach atomi in ATOMSLig do\n\nϕatoms = ProteinAtomsWithinRadiusOfLigandAtom(Protein,atomi, Radius);\n\nforeach atomj in ϕatoms do\n\nif atomi-atomj is in ϕAcceptedAtomPair then\n\nInsertInMotifSet(atomj, ϕmotif);\n\nif (ϕmotif == n) then\n\nlast ;\n\nend\n\nend\n\nend\n\n/* increment radius by 0.1 Å */\n\nRadius = Radius + 0.1;\n\nend\n\nreturn ϕmotif ;\n\nend\n\nEach position of the motif has a set of amino acids specified to allow for stereochemically equivalent matches at that particular position (Equation 3), such that while matching amino acid type of ri should belong to GROUPi.\n\n\n\nPreviously, the K sets of N residues were obtained in Ptarget using an exhaustive search procedure similar to the one used in SPASM18. An enhanced algorithm now precompiles all possible motifs of a set (N=4 in this case) of predefined amino acid residues from a protein structure that occur within a specified distance16, and selects the appropriate ones based on each motif (Equation 4). Any match below a user defined threshold score (Sthresh) is discarded.\n\n\n\nIn Case ΦmatchesPi is null, the ligand Lig can not be docked to the target protein Ptarget. M1Pi, the first element, has the minimum CScore and represents the putative binding site in Ptarget based on the holoenzyme Pi. The set of putative binding sites Φbindsite is thus defined (Equation 5).\n\n\n\nEach element of Φbindsite (M1Pi) is now superimposed to the corresponding holoenzyme, based on the motif binding Lig in Pi. In order to superimpose these motifs, linear and rotational transformations are applied on all atoms such that the first three atoms lie on the same plane (Z=0), the first atoms are the origin of the coordinate axis and the second atoms lie on the Y axis. This creates a unified coordinate framework having the holoenzyme, the ligand and the target enzyme, thus providing the docked ligand in the target enzyme. Essentially, the holoenzyme is replaced with the target enzyme if the contact points have a good spatial and electrostatic match in the target enzyme by aligning the congruent atoms. This docked ligand is now outputted as a Pymol formatted file.\n\nThe DOCLASP package is written in Perl on Ubuntu. Hardware requirements are modest - all results here are from a simple workstation (2GB ram) and runtimes were a few minutes at the most. Adaptive Poisson-Boltzmann Solver (APBS) and PDB2PQR packages were used to calculate the potential difference between the reactive atoms of the corresponding proteins19,20. The APBS parameters and electrostatic potential units were set as described previously in12. All protein structures were rendered by PyMol (http://www.pymol.org/).\n\n\nResults and discussion\n\nPrevious CLASP analysis of the spatial and electrostatic properties of active site residues in PI-PLC from B. cereus indicated that it is a prolyl peptidase, which was also validated by in vitro experiments13. Subsequently, it was shown that PI-PLC is inhibited by two dipeptidyl peptidase-IV (DPP4) inhibitors - vildagliptin (LAF-237) at micromolar concentrations, and K-579 at nanomolar concentrations. Since there are no DPP4 structures solved which ligand K-579, a DPP4 protein structure in complex with vildagliptin (PDBid:3W2TA)21 provided the five closest atoms in the protein (E205, E206, S630, Y662 and Y547) (see Methods) that make non-hydrophobic interactions with the ligand (Table 1).\n\nInteractions are sorted based on the distance. R/A/LA/D: Residue number/Atom of the residue/Atom of ligand/distance between the interacting atoms (in Å). For example, ‘S630/OG/N2/2.4’ means that the atom OG from Ser630 is at 2.4 Å from the N2 atom of vildagliptin in PDBid:3W2TA.\n\nA subset of these atoms might be sufficient to ligand vildagliptin in the target protein. Thus, 54=5 motifs, each with four atoms, were created using the five closest atoms in the protein. The binding of ligands is known to induce electrostatic and spatial perturbations in the binding site. The spatial and electrostatic perturbations induced by the vildagliptin binding is shown by comparing the apo (PDBid:2OQIA) and the holoenzyme (PDBid:3W2TA) in Table 2. Hence, the electrostatic and spatial profile of the motif were obtained from the apo DPP4 enzyme (PDBid:2OQIA), and then used for querying the PI-PLC apo structure (PDBid:1PTDA).\n\nWe compare the pairwise distance and electrostatic potential difference (EPD) changes in the apo (PDBid:2OQIA) and holo (PDBid:3W2TA) enzymes. Note that the pairwise distance between these atoms change in the ligand free PDB (2OQIA) as compared to the protein with bound inhibitor (2BUBA). For example, the distance between E205OE2 and E206OE2 (pair ab) changes from 3.9 Å to 5.6 Å. Also, there is a definite change in the EPD between E205OE2 and E205OE2 (pair ab). D = Pairwise distance in Å. PD = Pairwise potential difference. The electrostatic potential are in dimensionless units of kT/e where k is Boltzmann’s constant, T is the temperature in K and e is the charge of an electron.\n\nThese motifs were used to query the PI-PLC structure using an enhanced algorithm (PREMONITION) that precompiles all motifs in a database16. Table 3 shows the best matches obtained in the PI-PLC structure for the five partial motifs. All these matches have significant electrostatic congruence. The root mean square deviation (RMSD) have low values - however, this is a deceptive metric since these deviations are averaged out. The maximum pairwise distance is another metric to discriminate the spatial congruence, and should be used in combination with the RMSD value. All of these above mentioned matches have significant maximum pairwise distance deviation. Also, three matches do not comprise of active site residues (motifs 2, 3 and 4). However, it can be seen that the first and fifth matches comprises of active site residues (involved in the binding of myo-inositol in PDBid:1PTGA), and have three residues (Asp67, Asp198 and Trp178) in common.\n\nThe comparison is done using apo enzymes (PDBid:1PTDA for PI-PLC and PDBid:2OQIA for DPP4), since the binding of a ligand induces spatial and electrostatic changes in the active site. The fifth motif has the least rmsd deviation, and comprises of active site residues (involved in the binding of myo-inositol in PDBid:1PTGA). Out of the best matches in the other four motifs, three do not comprise of active site residues (motifs 2, 3 and 4). Motif 1 has a reasonably significant match, and has three residues (Asp67, Asp198 and Trp178) in common with the best match for Motif 5. These three residues from PI-PLC (Asp67, Asp198 and Trp178) and corresponding three residues from DPP4 (Glu205, Glu206 and Tyr662) were used to superimpose DPP4 and PI-PLC. N = Motif number. D = Pairwise distance in Å. PD = Pairwise potential difference. Rmsd = Root mean square deviation. Max = maximum pairwise distance deviation. APBS writes out the electrostatic potential in dimensionless units of kT/e where k is Boltzmann’s constant, T is the temperature in K and e is the charge of an electron.\n\nThus, these three residues from PI-PLC (Asp67, Asp198 and Trp178) and corresponding three residues from DPP4 (Glu205, Glu206 and Tyr662) were used to superimpose DPP4 and PI-PLC. Table 4 shows the congruence of these residues. The corresponding holo structures - PDBid:3W2TA for DPP4, and PDBid:1PTGA for PIPLC - were used for the superimposition. This superimposition applies geometric transformations such that Asp67OD1 and Glu205OE2 were at the center of the coordinate axis (coordinates = [0,0,0]), Asp198OD1 and Glu206OE2 lies on the X-Y axis (i.e. Y coordinate is 0) and Tyr662CZ and Trp178CZ2 were on the X-Y plane (i.e. Z coordinate is 0). Figure 1 shows the superimposed proteins. It is observed that (Asp67, Asp198 and Trp178) overlaps well with (Glu205, Glu206 and Tyr662).\n\nThe match is significant and comprises active site residues (involved in the binding of myo-inositol in PDBid:1PTGA). Pair ‘ab’ is considered to be electrostatically congruent since the PD values are close to zero, and can be considered almost equipotential. This is expected for atoms of the same type from the same residue (GLU205OE1/GLU206OE1 and ASP67OD1/ASP198OD1). These three residues from PI-PLC (Asp67, Asp198 and Trp178) and corresponding three residues from DPP4 (Glu205, Glu206 and Tyr662) were used to superimpose DPP4 and PI-PLC. D = Pairwise distance in Å. PD = Pairwise potential difference. APBS writes out the electrostatic potential in dimensionless units of kT/e where k is Boltzmann’s constant, T is the temperature in K and e is the charge of an electron.\n\nThree residues from PI-PLC (Asp67, Asp198 and Trp178 in yellow) were superimposed to the corresponding three residues from DPP4 (Glu205, Glu206 and Tyr662 in red). Asp67OD1 and Glu205OE2 is at the center of the coordinate axis (coordinates = [0,0,0]) (in black), Asp198OD1 and Glu206OE2 lies on the X-Y axis (i.e. Y coordinate is 0) and Tyr662CZ and Trp178CZ2 are on the X-Y plane (i.e. Z coordinate is 0). Asp67, Asp198 and Trp178 in the PI-PLC protein overlaps well with Glu205, Glu206 and Tyr662 from DPP4, but the Ser234-Ser630 pair is not spatially congruent.\n\nThese transformations were also applied to the vildagliptin molecule, and this resulted in a docked structure for this molecule into the PI-PLC protein. Figure 2 shows the vildagliptin docked into the PI-PLC structure which is complexed with myo-inositol (PDBid:1PTGA). The distances of the atoms in vildagliptin and myo-inositol that interact (excluding hydrophobic interactions) to the first ten residues in the PI-PLC structure are shown in Table 5. It is interesting to note that the residues shown in Table 5 are all part of side chain residues in close contact with the myo-inositol ring (shown in Figure 713). Further validation was obtained by observing that both Arg69/NH1 and His32/NE2 interact with atom O4 in vildagliptin, and Arg69/NH2 and His32/NE2 interact with O2 in myo-inositol in the PI-PLC structure. The Pymol script for visualizing the docking (SupplementaryPymol.p1m) and a movie (SupplementaryMovie.avi) are also provided as Supplementary information.\n\nIt can be seen that vildagliptin fits into the binding site of PI-PLC. It also makes non-hydrophobic contacts to the residues in the protein similar to those made by myo-inositol (Table 5).\n\nThese interactions exclude hydrophobic interactions, and the first closest ten atoms are chosen. Out of ten, seven residues obtained by docking vildagliptin using DOCLASP are seen to be equivalent to those that are known to bind myo-inositol to the PI-PLC structure13, while two more have the same amino acid type (marked by asterisks). Only one pair has a different amino acid type (Tyr200 for myo-inositol and Glu117 for vildagliptin). Further validation is obtained by observing that both Arg69/NH1 and His32/NE2 both interact with atom O4 in vildagliptin, and Arg69/NH2 and His32/NE2 interact with O2 in myo-inositol in the PI-PLC structure.\n\nIt is important to comment on the previous hypothesis of a nucleophilic serine being responsible for the inhibition of PI-PLC using vildagliptin. The electrostatic and spatial profile of the motif 4 from DPP4 is compared to the electrostatic and spatial profile of matching active site residues in PI-PLC in Table 6, including serine in the comparison. It can be seen that Ser630 in DPP4 has a significant spatial difference as compared to Ser234 in PI-PLC (pair ‘bc’ has a difference of 5 Å), and also a reasonable electrostatic difference (pair ‘ac’ has a difference of 144 PD units). The relatively large distance over which Ser234 in PI-PLC interacts with myo-inositol (4.8 Å) indicates that Ser234 is not directly involved in the binding of the ligand. However, it is responsible for creating the electrostatic milieu that is required for other interacting residues to attain their appropriate potential. Even for DPP4, many inhibitors do not interact with the nucleophilic Ser630 (manuscript in preparation) - although the vildagliptin molecule does21. Thus, the previous conjecture of a nucleophilic serine being directly responsible for the binding of DPP4 inhibitors to PI-PLC, as implied by the catalytic triad congruence, is incorrect13. However, this serine is indirectly responsible for driving the neighboring residues to an appropriate state. Spatial constraints are an additional discriminator.\n\nBoth the structures are apo enzymes, since the binding of a ligand induces spatial and electrostatic changes in the active site. Ser630 in DPP4 has a significant spatial difference as compared to Ser234 in PI-PLC (pair ‘bc’ has a difference of 5 Å), and also a reasonable electrostatic difference (pair ‘ac’ has a difference of 144 PD units). D = Pairwise distance in Å. PD = Pairwise potential difference. APBS writes out the electrostatic potential in dimensionless units of kT/e where k is Boltzmann’s constant, T is the temperature in K and e is the charge of an electron.\n\nDOCLASP was also used recently22 to dock human karyopherin to the VP2423 protein of the Reston Ebola strain using the VP24 from Zaire Ebola24 as a template, and demonstrate that a single mutation might be one of the critical factors responsible for the non-pathogenic nature of Reston Ebola in humans25,26.\n\nThe number of docking methods available is such that even a detailed review could only provide a partial list of currently available docking methods9. The current work presents a template based, static method that leverages the spatial and electrostatic properties of the binding site. The definitive advantage of a static method can only be highlighted by emphasizing the known limitations of conformational sampling of the protein structure27,28. The problem is indeed exacerbated by the plasticity of the drug itself29–31. While DOCLASP is completely ineffectual in the absence of such a database, unlike de novo methods, it benefits from the burgeoning database of protein-ligand structures32. The conservation of electrostatic properties, extracted using APBS/PDB2PQR, is the strongest argument in favor of DOCLASP.\n\nThere are several limitations in the method. Firstly, it can be applied to those compounds which are bound to proteins whose structures have been solved. Additionally, it is requires the structure of the apoenzyme, as this is used to extract the query motif considering the structural and electrostatic changes induced by ligand binding. However, with an ever increasing number of protein structures being solved, this is not a severe limitation since most proteins with ligands also have their apo structures solved. Furthermore, the lack of congruent matches leads DOCLASP to return a null result. This can be overcome by relaxing constraints, for example by checking for spatial congruence only. Finally, it is required to develop an energy function which will be able to discriminate poorly docked structures that have either significant steric clashes or are docked on the surface of the protein.\n\nTo summarize, this work presents an implicit method for docking ligands to proteins, in which the search and scoring are implicit in the CLASP algorithm. One significant limitation of this method is the requirement of template protein structures in complex with the given compound. As future work, I intend to incorporate the flexibility of the ligand and protein to add further discrimination.",
"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\nSupplementary materials\n\nPymol script for visualizing the docking and movie.\n\nThe file “SupplementaryPymol.p1m” contains the Pymol file for viewing vildagliptin docked to PIPLC and the file “SupplementaryMOvie.avi” contains a movie showing a 360 rotation of the ligand docked to PIPLC.\n\nClick here to access the data.\n\n\nReferences\n\nCosconati S, Forli S, Perryman AL, et al.: Virtual Screening with AutoDock: Theory and Practice. Expert Opin Drug Discov. 2010; 5(6): 597–607. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTanrikulu Y, Kruger B, Proschak E: The holistic integration of virtual screening in drug discovery. Drug Discov Today. 2013; 18(7–8): 358–364. PubMed Abstract | Publisher Full Text\n\nSeddon G, Lounnas V, McGuire R, et al.: Drug design for ever, from hype to hope. J Comput Aided Mol Des. 2012; 26(1): 137–150. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorris GM, Huey R, Lindstrom W, et al.: AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem. 2009; 30(16): 2785–2791. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones G, Willett P, Glen RC, et al.: Development and validation of a genetic algorithm for flexible docking. J Mol Biol. 1997; 267(3): 727–748. PubMed Abstract | Publisher Full Text\n\nEwing TJ, Makino S, Skillman AG, et al.: DOCK 4.0: search strategies for automated molecular docking of flexible molecule databases. J Comput Aided Mol Des. 2001; 15(5): 411–428. PubMed Abstract | Publisher Full Text\n\nSchellhammer I, Rarey M: FlexX-Scan: fast, structure-based virtual screening. Proteins. 2004; 57(3): 504–517. PubMed Abstract | Publisher Full Text\n\nFriesner RA, Banks JL, Murphy RB, et al.: Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem. 2004; 47(7): 1739–1749. PubMed Abstract | Publisher Full Text\n\nSousa SF, Ribeiro AJ, Coimbra JT, et al.: Protein-ligand docking in the new millennium--a retrospective of 10 years in the field. Curr Med Chem. 2013; 20(18): 2296–2314. PubMed Abstract | Publisher Full Text\n\nGrinter SZ, Zou X: Challenges, applications, and recent advances of protein-ligand docking in structure-based drug design. Molecules. 2014; 19(7): 10150–10176. PubMed Abstract | Publisher Full Text\n\nYuriev E, Agostino M, Ramsland PA: Challenges and advances in computational docking: 2009 in review. J Mol Recognit. 2011; 24(2): 149–164. 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\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\nChakraborty S, Rendon-Ramirez A, Asgeirsson B, et al.: Dipeptidyl peptidase-iv inhibitors used in type-2 diabetes inhibit a phospholipase c: a case of promiscuous scaffolds in proteins [v1; ref status: approved 1, approved with reservations 1, http://f1000r.es/2hw]. F1000Res. 2013; 2: 286. Publisher Full Text\n\nChakraborty S, Rao BJ, Asgeirsson B, et al.: Premonition - preprocessing motifs in protein structures for search acceleration [v1; ref status: awaiting peer review, http://f1000r.es/492]. F1000Res. 2014; 3: 217. Publisher Full Text\n\nHeinz DW, Ryan M, Bullock TL, et al.: Crystal structure of the phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with myo-inositol. EMBO J. 1995; 14(16): 3855–3863. PubMed Abstract | Free Full Text\n\nKleywegt GJ: Recognition of spatial motifs in protein structures. J Mol Biol. 1999; 285(4): 1887–1897. PubMed Abstract | Publisher Full Text\n\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\nDolinsky TJ, Nielsen JE, McCammon JA, et al.: PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 2004; 32(Web Server issue): W665–667. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNabeno M, Akahoshi F, Kishida H, et al.: A comparative study of the binding modes of recently launched dipeptidyl peptidase IV inhibitors in the active site. Biochem Biophys Res Commun. 2013; 434(2): 191–196. PubMed Abstract | Publisher Full Text\n\nChakraborty S, Rao B, Asgeirsson B, et al.: Correlating the ability of VP24 protein from Ebola and Marburg viruses to bind human karyopherin to their immune suppression mechanism and pathogenicity using computational methods [v1; ref status: awaiting peer review, http://f1000r.es/4o3]. F1000Res. Publisher Full Text\n\nZhang AP, Abelson DM, Bornholdt ZA: The Ebolavirus VP24 interferon antagonist: Know your enemy. Virulence. 2012; 3(5): 440–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXu W, Edwards MR, Borek DM: Ebola virus VP24 targets a unique NLS binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated STAT1. Cell Host Microbe. 2014; 16(2): 187–200. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiranda ME, White ME, Dayrit MM: Seroepidemiological study of filovirus related to Ebola in the Philippines. Lancet. 1991; 337(8738): 425–426. PubMed Abstract | Publisher Full Text\n\nMiranda ME, Miranda NL: Reston ebolavirus in humans and animals in the Philippines: a review. J Infect Dis. 2011; 204(8738): S757–S760. PubMed Abstract | Publisher Full Text\n\nDurrant JD, McCammon JA: Molecular dynamics simulations and drug discovery. BMC Biol. 2011; 9: 71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBorhani DW, Shaw DE: The future of molecular dynamics simulations in drug discovery. J Comput Aided Mol Des. 2012; 26(1): 15–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPerola E, Charifson PS: Conformational analysis of drug-like molecules bound to proteins: an extensive study of ligand reorganization upon binding. J Med Chem. 2004; 47(10): 2499–2510. PubMed Abstract | Publisher Full Text\n\nSturm N, Desaphy J, Quinn RJ, et al.: Structural insights into the molecular basis of the ligand promiscuity. J Chem Inf Model. 2012; 52(9): 2410–2421. PubMed Abstract | Publisher Full Text\n\nMobley DL, Dill KA: Binding of small-molecule ligands to proteins: “what you see” is not always “what you get”. Structure. 2009; 17(4): 489–498. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu T, Lin Y, Wen X, et al.: BindingDB: a web-accessible database of experimentally determined protein–ligand binding affinities. Nucl Acids Res. 2007; 35(Database issue): D198–D201. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "7194",
"date": "20 Jan 2015",
"name": "Rahul Banerjee",
"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 has presented a novel docking algorithm based on the principle that spatial and electrostatic properties in cognate pairs of residues are conserved in the catalytic site of proteins endowed with the same function. Thus, in case the crystal structure of a protein-ligand complex is available, it can be used to dock the same ligand into the active site of a homologous protein (with identical catalytic activity). The author has tested the algorithm by docking vildagliptin into the active site of PI-PLC.Examination of Figure 2 and Table 5 show quite a few short contacts between the docked ligand and amino acid chains. Under such circumstances the author could consider the inclusion of an energy minimization protocol to relieve the short contacts. The author considers the method validated by the above mentioned docking exercise. However, the author could consider a case where the actual crystal structure of the solution exists and the rmsd between the experimental solution and the solution obtained by DOCLASP could be provided to give some estimate of the accuracy of the docked poses.Given the fact that the DOCLASP solution abounds in short contacts, further improvements could be made in the pose. On page 3, two lines below Equation 4, a more explicit account of CScore could be given in the manuscript.",
"responses": [
{
"c_id": "2012",
"date": "16 Jun 2016",
"name": "Sandeep Chakraborty",
"role": "Author Response",
"response": "Dear Dr Banerjee, I would like to thank you for taking the time to review this paper, and for your positive comments. I also apologize for the time taken to respond to your comments, a delay that took place due to the time taken to find another reviewer. Please find my detailed responses to your comments below. The author has presented a novel docking algorithm based on the principle that spatial and electrostatic properties in cognate pairs of residues are conserved in the catalytic site of proteins endowed with the same function. Thus, in case the crystal structure of a protein-ligand complex is available, it can be used to dock the same ligand into the active site of a homologous protein (with identical catalytic activity). The author has tested the algorithm by docking vildagliptin into the active site of PI-PLC. 1. Examination of Figure 2 and Table 5 show quite a few short contacts between the docked ligand and amino acid chains. Under such circumstances the author could consider the inclusion of an energy minimization protocol to relieve the short contacts. 2. The author considers the method validated by the above mentioned docking exercise. However, the author could consider a case where the actual crystal structure of the solution exists and the RMSD between the experimental solution and the solution obtained by DOCLASP could be provided to give some estimate of the accuracy of the docked poses. Given the fact that the DOCLASP solution abounds in short contacts, further improvements could be made in the pose. Since, DOCLASP uses templates from holoenzymes, re-liganding them back would be trivially correct all the time. Energy minimizations would certainly help in resolving the steric constraints. Such methods are standard, and can be applied to the output PDB of DOCLASP, but have not been done in the current version. I will consider including this in the future. Instead, I have included two additional test cases, DOCLASP was previously applied to the binding of phenylthiourea to polyphenol oxidases (PPO) from walnut (JrPPO1) (http://onlinelibrary.wiley.com/doi/10.1111/tpj.13207/). Incidentally, the structure of this protein was solved while the walnut genome manuscript was in review (PDBid:5CE9). Here, phenylthiourea was docked to this solved structure, resulting in almost the same pose as the one in which DOCLASP docked phenylthiourea to the SWISSMODEL-modelled structure of JrPPO1. Secondly, a comprehensive analysis of the binding of suramin to different non-homologous proteins from the PDB database has been presented, and DOCLASP was used to dock suramin to a phospholipase A2-like protein. This highlights the non-specific binding of some ligands, and also the complexity of modelling a flexible ligand like suramin. 3. On page 3, two lines below Equation 4, a more explicit account of CScore could be given in the manuscript. I have modified the methods section to give a more explicit explanation of CScore. I appreciate your consideration of the revised manuscript, and look forward to hearing back from you. best wishes, Sandeep"
}
]
},
{
"id": "13760",
"date": "24 May 2016",
"name": "Amarda Shehu",
"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 serves as a proof of concept. It tests the idea, previously published, that if spatial and electrostatic similarities between cognate pairs of residues are predictive of catalytic function, then aligning two proteins to match 4 cognate pairs of residues ought to identify the binding site in a novel/target protein. The alignment process additionally provides the pose of the ligand in the target protein, if the matching is conducted with a holoenzyme.\nAs a proof of concept, the article is scientifically sound.\nMajor observations:\nIt would be desirable to provide more testing sets. That is, expand the evaluation to more than binding of vildagliptin to PI-PLC. It is trivial to do so. The authors can use known protein-ligand complex cases, treat them as unknown effectively, and test whether they are able to reproduce the binding pose for the ligand.\n\nExpanding the test set will also improve the evaluation of the proposed DOCLASP protocol. The selected case study is presented in detail, but how would one determine the effectiveness of the method at a large scale? What would be summary results on application of DOCLASP on a set of test cases?\n\nThe author states that a limitation of the method, as a template-based one, is the availability of protein-ligand pairs with known structures. Given the growth in structural data, this is bound to be less of a limitation now as opposed to ten years ago. However, what needs to be discussed is the sensitivity of the method. Is the author reliant on structures with good resolution? What would this be? If an X-ray structure has 2.5A or lower resolution as opposed to 1.5, should the results be trusted less?\n\nFollowing on above, if two different poses are obtained by matching with two different holoenzymes, and one of the holoenzymes has resolution 2.5 but the other has resolution 1.5, should this be considered in what pose is recommended for the ligand in the binding protein? What is the reliance of predictions on quality of input?\n\nIn case of pose discrepancies from diversity of quality of holoenzymes, would energetic minimization help converge to the right pose? This would be an interesting experiment to conduct. In particular, the experiment can suggest that a specific threshold in quality is needed for the minimization to converge to the right pose.\nMinor:\nConsider using a different symbol to denote the number of holoenzymes, M, from the symbol used to denote motif, M. In particular, this can cause confusions regarding equation (5). P1...PM relates to the set of holoenzymes, whereas M1^{PM} relates to best-scoring motif extracted from the last holo-enzyme, PM.\n\nGiven that there are M holoenzymes, it is unclear whether the method produces M possible poses for the ligand, or if these poses are filtered or joined in some way to provide a super pose.\n\nConsider using passive form rather than active \"I\" form when summarizing future work.",
"responses": [
{
"c_id": "2013",
"date": "16 Jun 2016",
"name": "Sandeep Chakraborty",
"role": "Author Response",
"response": "Dear Dr Shehu, I would like to thank you for taking the time to review this paper, and providing constructive criticism on the overall manuscript. I have revised the manuscript keeping these in mind. Below I outline a point-by-point response to your comments. This article serves as a proof of concept. It tests the idea, previously published, that if spatial and electrostatic similarities between cognate pairs of residues are predictive of catalytic function, then aligning two proteins to match 4 cognate pairs of residues ought to identify the binding site in a novel/target protein. The alignment process additionally provides the pose of the ligand in the target protein, if the matching is conducted with a holoenzyme. As a proof of concept, the article is scientifically sound. I greatly appreciate the positive comment. Major observations: 1. It would be desirable to provide more testing sets. That is, expand the evaluation to more than binding of vildagliptin to PI-PLC. It is trivial to do so. The authors can use known protein-ligand complex cases, treat them as unknown effectively, and test whether they are able to reproduce the binding pose for the ligand. Since, DOCLASP uses templates from holoenzymes, re-liganding them back would be trivially correct all the time. However, I have added two testcases. The first docks phenylthiourea to polyphenol oxidases (PPO) from walnut (JrPPO1). In a recent paper on the walnut genome sequence (http://onlinelibrary.wiley.com/doi/10.1111/tpj.13207/), DOCLASP was used to dock phenylthiourea to the modelled structure of JrPPO1. Incidentally, the structure of this protein was solved while the walnut genome manuscript was in review (PDBid:5CE9). In the current work, docking phenylthiourea to this structure resulted in almost the same pose. Secondly, a comprehensive analysis of the binding of suramin to different non-homologous proteins from the PDB database has been presented, and DOCLASP was used to dock suramin to a phospholipase A2-like protein. This highlights the non-specific binding of some ligands, and also the complexity of modelling a flexible ligand like suramin. 2. Expanding the test set will also improve the evaluation of the proposed DOCLASP protocol. The selected case study is presented in detail, but how would one determine the effectiveness of the method at a large scale? What would be summary results on application of DOCLASP on a set of test cases? A large scale analysis is desirable, but difficult for an individual independent researcher. Fortunately, the f1000 allows theoretically infinite revisions. This will be work in progress. 3. The author states that a limitation of the method, as a template-based one, is the availability of protein-ligand pairs with known structures. Given the growth in structural data, this is bound to be less of a limitation now as opposed to ten years ago. However, what needs to be discussed is the sensitivity of the method. Is the author reliant on structures with good resolution? What would this be? If an X-ray structure has 2.5A or lower resolution as opposed to 1.5, should the results be trusted less? 4. Following on above, if two different poses are obtained by matching with two different holoenzymes, and one of the holoenzymes has resolution 2.5 but the other has resolution 1.5, should this be considered in what pose is recommended for the ligand in the binding protein? What is the reliance of predictions on quality of input? Empirically, I would assume that the sensitivity of this method would improve with lower resolution, but I do not know how to establish that scientifically. DOCLASP also depends on APBS for computing electrostatic potential, and that is known to improve with lower resolution. 5. In case of pose discrepancies from diversity of quality of holoenzymes, would energetic minimization help converge to the right pose? This would be an interesting experiment to conduct. In particular, the experiment can suggest that a specific threshold in quality is needed for the minimization to converge to the right pose. Energetic minimization would certainly help in resolving steric clashes. Since there are several well established methods that can be applied to the output PDB from DOCLASP, I have not discussed these in the current paper. Minor: 1. Consider using a different symbol to denote the number of holoenzymes, M, from the symbol used to denote motif, M. In particular, this can cause confusions regarding equation (5). P1...PM relates to22 the set of holoenzymes, whereas M1PM relates to best-scoring motif extracted from the last holo-enzyme, PM. Done (number of holoenzymes is denoted by Z). 2. Given that there are M holoenzymes, it is unclear whether the method produces M possible poses for the ligand, or if these poses are filtered or joined in some way to provide a super pose. If there are multiple holoenzymes, DOCLASP will generate a pose for each (assuming there is a significant match of the motif from the holoenzyme). Currently, there is no method to create a single ’super pose’ from these. 3. Consider using passive form rather than active ”I” form when summarizing future work. Done. I appreciate your consideration of the revised manuscript, and look forward to hearing back from you. best wishes, Sandeep"
},
{
"c_id": "2037",
"date": "21 Jun 2016",
"name": "Amarda Shehu",
"role": "Reviewer Response",
"response": "I will be keen to see the progress of this work, particularly with regards to an integrative setting that considers various structural models and further interface with energetic minimization protocols. I do sympathize with the comment on an independent researcher aiming to share research progress in spite of large-scale evaluation settings. F1000 is the right venue for these types of dissemination."
}
]
}
] | 2
|
https://f1000research.com/articles/3-262
|
https://f1000research.com/articles/5-1370/v1
|
14 Jun 16
|
{
"type": "Review",
"title": "Yersinia virulence factors - a sophisticated arsenal for combating host defences",
"authors": [
"Steve Atkinson",
"Paul Williams"
],
"abstract": "The human pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica cause enterocolitis, while Yersinia pestis is responsible for pneumonic, bubonic, and septicaemic plague. All three share an infection strategy that relies on a virulence factor arsenal to enable them to enter, adhere to, and colonise the host while evading host defences to avoid untimely clearance. Their arsenal includes a number of adhesins that allow the invading pathogens to establish a foothold in the host and to adhere to specific tissues later during infection. When the host innate immune system has been activated, all three pathogens produce a structure analogous to a hypodermic needle. In conjunction with the translocon, which forms a pore in the host membrane, the channel that is formed enables the transfer of six ‘effector’ proteins into the host cell cytoplasm. These proteins mimic host cell proteins but are more efficient than their native counterparts at modifying the host cell cytoskeleton, triggering the host cell suicide response. Such a sophisticated arsenal ensures that yersiniae maintain the upper hand despite the best efforts of the host to counteract the infecting pathogen.",
"keywords": [
"Yersinia pestis",
"yersiniae",
"ail locus",
"pH6 antigen",
"virulence factors"
],
"content": "Introduction\n\nAcross an infection timeline, the host and invading bacterial pathogen each vie for supremacy. At any given time, either may have the upper hand, but the final outcome of this battle ultimately determines the fate of the host. The triggered host response will aim to reduce the infectivity of the pathogen, but in order to stay one step ahead many bacterial species have evolved sophisticated strategies to ensure they can successfully cause infection following colonisation.\n\nThe three human pathogens belonging to the genus Yersinia employ a range of virulence factors that confer efficient adherence to host cells/tissues and subvert host cell functions. This mini-review highlights the key virulence factors that constitute the virulence arsenal of Yersinia spp. and how such a sophisticated suite of biological weapons enables these pathogens to combat host defences.\n\nYersinia pseudotuberculosis, Yersinia pestis, and Yersinia enterocolitica are highly adaptable psychrotrophic primary human pathogens. Y. pseudotuberculosis and Y. enterocolitica cause self-limiting gastric infections. Y. pestis is a recently evolved near-identical subclone of Y. pseudotuberculosis1,2 with approximately 98% identity at the DNA level. Its strategy for transmission relies on the colonisation of rat fleas, which then carry Y. pestis between the rodent host and humans3. Once inside the human host, Y. pestis can cause bubonic, pneumonic, and septicaemic plague with mortality rates approaching 100% without antibiotic treatment4. The World Health Organisation considers Y. pestis a ‘re-emerging’ pathogen that, worryingly, is capable of acquiring resistance to multiple antibiotics5 and is also a serious potential bioterrorism threat. The differences in lifestyle and virulence between Y. pseudotuberculosis and Y. pestis are mostly attributable to minor genomic differences on the respective chromosomes and the presence of two additional virulence plasmids that Y. pestis possesses.\n\n\nThe Yersinia type three secretion system\n\nThe key Yersinia virulence determinants and certainly the most comprehensively studied are those secreted via a type three secretion system (T3SS). To evade host innate immunity and to enable the pathogen to replicate and propagate extracellularly, all human pathogenic Yersinia species harbour an approximately 70 kb virulence plasmid. Located on this plasmid is a set of genes whose transcription is activated by temperatures of 37°C in the presence of millimolar concentrations of calcium, conditions representing the mammalian host. These genes code for the T3SS ‘nanomachine’, a hypodermic needle-like structure (the injectisome) and the translocon, which forms a pore across the host cell membrane (Figure 1). Along with a combination of regulators and chaperones, the T3SS’s primary function is to inject multiple toxic Yersinia effector proteins (Yops) directly into the eukaryotic host cell cytosol. Once inside, they subvert host cell signalling pathways and trigger a pre-programmed metabolic chain reaction that results in apoptosis6,7. Yops also inhibit phagocytosis and block cytokine production.\n\nThe needle is fixed into the bacterial inner and outer membrane and protrudes from the surface to penetrate the host membrane. The translocon forms a channel through the host membrane and the Yop effectors are transferred into the host from the bacterial cytoplasm via the needle and translocon (a). The needle protrudes from the bacterial surface prior to host cell penetration (b, c arrowed). Salmonella typhimurium T3SS needles isolated from the bacterial membrane (d). (a) adapted from 133, (b) reproduced with permission and taken from reference 56, (c) reproduced with permission and taken from reference 16, and (d) reproduced from reference 134.\n\n\nThe structure of the T3SS needle and translocon\n\nStructurally, the base of the injectisome is composed of a number of proteins that adopt a cylindrical architecture similar to that of the flagellar basal body8 that are directed to the membrane by the secretion (Sec)-dependent pathway9. The injectisome incorporates two membrane rings termed the MS (membrane and supramembrane) and OM (outer membrane) rings. These are connected to five integral membrane proteins that play a role in exporting proteins10,11 (Figure 1). The export apparatus itself is flanked by YscQ, which facilitates the binding of the ATPase YscN and the secretion substrate-chaperone complexes12. YscN provides the proton motive force necessary for driving the secretion of the Yop effectors9,13,14.\n\nProtruding into the extracellular space from the basal body is a hollow needle formed by the helical polymerisation of YscF protein subunits9,15,16. YscF is exported and polymerised in a T3SS-dependent manner along with YscP, a protein akin to a molecular ruler that determines the length of the needle and limits its size17–19. It has recently been shown that fully formed T3SS needles form clusters on the bacterial cell surface and new needles appear to localise to these clusters rather than being randomly distributed20 (Figure 2). The needle tip is capped with LcrV21,22, a protein that directs the formation of a pore or ‘translocon’23. The translocon consists of a tripartite protein pore, which is inserted into host cell membranes and drives the translocation of Yop effectors into the host target cell cytoplasm. The pore is composed of the transmembrane proteins YopB and YopD23 and the injectisome tip complex LcrV24–26. Bacteria lacking the tip and translocon proteins are able to secrete effectors into the extracellular environment but are defective in translocating Yops into host cells27–29.\n\nReproduced from reference 20.\n\n\nChaperones facilitate the formation and operation of the T3SS\n\nGiven the complexity of the T3SS, part of its sophistication relates to its in-built ability to discriminate between structural and secretion substrates, providing strict order to ensure the needle is assembled and polymerised before translocon and Yop effector secretion30. Such ordering requires specific chaperones, typically small protein dimers that protect the target T3SS protein from degradation31,32 and prevent premature oligomerisation24 and also ushering into the injectisome. These T3SS chaperones are usually subdivided into three classes: class I chaperones bind the Yop effector proteins and often share high structural conservation, class II chaperones associate with the translocon proteins YopB, YopD, and LcrV, and class III chaperones tend to form heterodimers and associate with structural components of the injectisome.\n\n\nThe Yop effectors\n\nThe Yop effector proteins are virulence factors synthesised in the bacterial cytoplasm and secreted through the T3SS needle and translocon into eukaryotic target cells (Figure 1). Four of these (YopE, YopT, YpkA, and YopH) are involved in disrupting the normal activities of the cytoskeleton and, apart from YopH, also target an important group of eukaryotic cell signalling components, the RhoA family of small GTPases that direct cytoskeletal rearrangements necessary for phagocytosis. YopE is a functional mimic of eukaryotic GTPase-activating proteins (GAPs)33 and disrupts the actin cytoskeleton34–36, resulting in the inhibition of phagocytosis by macrophages. YopT suppresses RhoA-mediated signalling by cleaving the post-translationally modified Rho GTPases37, which ultimately prevents the formation of the phagocytic cup for bacterial internalisation, and inhibits the assembly of focal adhesion complexes required for the development of pseudopodia and macrophage migration38,39. YpkA (YopO in Y. enterocolitica) associates with RhoA family proteins40,41 and inhibits phagocytosis42,43 by binding to and phosphorylating actin that is used as bait by Y. enterocolitica to titrate out host regulators responsible for actin polymerisation44. YopH is multi-functional and disrupts pathways involved in both innate and adaptive immunity and is essential for the virulence of Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica in mice45–47. YopH inhibits autophagy following binding of invasin or YadA (see next section) to β1-integrins48 and also blocks phagocytosis in macrophages49,50 by dephosphorylating focal adhesion complex proteins, which disrupts the link to the actin cytoskeleton51,52.\n\nThe remaining two effectors (YopJ and YopM) down-regulate elements of the immune system, such as inflammation and leukocyte recruitment53–57. YopJ (YopP in Y. enterocolitica) is a serine/threonine/lysine acetyltransferase that catalyses the acylation of kinases, inhibiting their ability to activate the release of NF-Κβ, which would otherwise induce pro-inflammatory cytokine production58–62. Recently, YopJ was also shown to play an important role in inhibiting caspase-1 in activated macrophages63.\n\nYopM is translocated into macrophages64 and may also be able to self-deliver into some human cells65, yet it has no known enzymatic activity66 and its true function has yet to be elucidated. Inside eukaryotic cells, YopM may interact with and stimulate cellular kinases67 and is thought to localise to the nucleus68–70, where it may influence the expression of a range of genes, down-regulating many pro-inflammatory cytokines65,71, counteract the innate immune system by promoting depletion of natural killer cells in the liver, spleen, and blood72, and also prevent pyroptosis by binding to caspase-1, inhibiting its activity73,74.\n\n\nYersinia surface adhesins\n\nFor yersiniae to efficiently deliver Yops into the host, it is essential that they adhere to the host cell surface and remain in close association during the delivery process. To ensure that this is possible, the yersiniae produce virulence factors in addition to the T3SS. An active T3SS can deliver effector proteins into the host cell cytosol only if the bacterial cells make direct contact with, and bind tightly to, the host cell surface. Over the last 30 years, several chromosomally or plasmid-encoded protein virulence factors have been identified that play a variety of roles in host cell attachment prior to effector protein injection. In each case, attachment is not their exclusive function and not all are present or active in all three of the human pathogens. However, a combination of these proteins confers the ability to adhere to and invade host cells or bind sufficiently to ensure successful T3SS delivery of Yops.\n\n\nInvasin\n\nInvasin is a chromosomally encoded protein that mediates attachment to and entry into host cells by Y. pseudotuberculosis and Y. enterocolitica75, although in Y. pestis it is a pseudogene and therefore inactive76 (Figure 3). Invasin promotes small intestine epithelial cell internalisation by binding to host cell target receptors known as β1-integrins77 that present on the host cell surface. Integrins form clusters upon invasin binding, and the result is the rearrangement of the host cell cytoskeleton. This promotes phagocytosis and ultimately internalisation of the bacteria into the epithelial cells. In fact, invasin has a significantly greater (up to 100 times) affinity for some integrins than its natural ligand, fibronectin78, and such strong associations are believed to be major contributing factors to the efficiency of internalisation and Yop delivery into host cells.\n\nVirulence factors found on the surface of Yersinia pseudotuberculosis, Yersinia enterocolitica (a), and Yersinia pestis (b) Ail, YadB, and YadC are shared by all three pathogens – YadB and YadC are absent from panel (a) for clarity – while Pla is unique to Y. pestis. YadA and invasin are important adhesins in Y. pseudotuberculosis and Y. enterocolitica but are not expressed by Y. pestis. Reproduced from reference 95.\n\nInvasin expression is regulated by both temperature and pH in Y. enterocolitica79,80. The invasin gene is maximally expressed at 26°C, peaking during late exponential/early stationary phase with lower expression levels observed at 37°C. This apparent contradiction, since invasin is required for infection at 37°C, was resolved when Pepe et al. revealed that the expression of invasin at 37°C was restored to levels seen at 26°C when the pH was reduced to 5.5. It has been suggested that rather than an experimental artefact, the expression of invasin at ambient temperatures could prepare the bacteria for infection following ingestion and promote rapid transcytosis through the epithelia81,82. The pH effect is not evident in Y. pseudotuberculosis, suggesting that the mechanisms of regulation of invasin expression may differ between the two species83. Two regulators have been found to be important for invasin expression: RovA, required for the positive regulation of invasin, and YmoA, required for negative regulation83–85. Both RovA and YmoA recognise the promoter region of invasin and compete for binding. Once RovA is bound, it appears to prevent YmoA from binding, thus inhibiting negative regulation of invasin86,87. The expression of rovA is itself regulated by temperature via RovM, which acts as a repressor of rovA expression under inducing growth conditions88.\n\n\nYadA\n\nAfter crossing the intestinal epithelium, the major adhesin responsible for Yersinia contact with cells of the submucosa is the virulence plasmid-encoded protein YadA (recently reviewed by Mühlenkamp et al.89) (Figure 3). YadA expression is induced at or above 37°C90,91, and under these conditions it is so abundant that it can virtually coat the entire outer surface of the bacterial cell92. Interestingly, despite YadA’s utility and abundance, Y. pestis possesses an inactive yadA pseudogene due to a single nucleotide deletion that results in a frame-shift mutation93 (Figure 3). Although Y. pestis does not produce a functional YadA protein, the chromosome carries two orthologues, YadB and YadC. Also found in Y. pseudotuberculosis, these two proteins are not thought to play a role in adherence but may contribute to host cell invasion. They may also be required for full virulence and lethality in bubonic but not pneumonic plague in mouse infection models94.\n\nYadA is a non-fimbrial adhesin95 belonging to the trimeric autotransporter adhesin family members, which are usually referred to as obligate homotrimeric proteins. The protein is shaped like a lollipop, with an N-terminal globular head domain connected by a coiled-coil stalk to a C-terminal anchor domain embedded in the outer membrane92. YadA has multiple functions but as an adhesin may act as a docking system, allowing the injectisome of the T3SS to come into contact with the target cell membrane to deliver the Yop effector proteins96.\n\nUntil recently, it was thought that YadA bound only to the large proteins of the extracellular matrix – collagen, fibronectin, and laminin – which in turn bind β1-integrins97–99. However, Keller et al.100 recently discovered that YadA-mediated adhesion may be facilitated by a broad range of host cell receptors and in the absence of β1-integrins may facilitate Yop injection via αV integrins as well as other unidentified cofactors. Y. enterocolitica YadA also binds leukocytes in a β1-integrin-independent manner during systemic infection101, all of which suggests that YadA has the potential to target a broad range of cell types to ensure efficient Yop delivery.\n\nThe collagen-binding activity of YadA in Y. enterocolitica is an absolute requirement for pathogenicity; however, YadA is not essential for virulence in Y. pseudotuberculosis97. YadA mediates adhesion to a number of cell types, including epithelial cells and macrophages, and can act as a haemagglutinin97. In Y. pseudotuberculosis, YadA promotes the invasion of epithelial cells and is interchangeable with the activity of invasin102, although Y. enterocolitica YadA is not as efficient an invasin as that of Y. pseudotuberculosis103. YadA also mediates bacteria-bacteria autoagglutination, since the head domain has an affinity for itself92. This self-affinity also promotes the formation of densely packed microcolonies that may promote antiphagocytic activity in Y. enterocolitica. YadA also binds to intestinal mucus104 and plays a major role in conferring serum resistance105–107.\n\n\nAil\n\nThe ail locus is chromosomally located and encodes a 17 kDa surface-associated protein (Figure 3) that is thermally regulated, being maximally expressed at 37°C108,109. In Y. enterocolitica, Ail-directed adhesion to host cells shows more specificity than invasin, as it allows invasion of some cell lines, such as human laryngeal epithelial type 2 (HEp-2), human endometrial (HEC-1B), and Chinese hamster ovary (CHO) cells, but no invasion of Madin-Darby canine kidney (MDCK) cells110. Both laminin and fibronectin are known targets for Y. pestis Ail binding111 and vitronectin is actively recruited to the Y. pestis surface through the activities of Ail112. Interestingly, Y. pseudotuberculosis Ail is unable to promote the attachment and invasion phenotypes when expressed in Escherichia coli113. As with invasin, Ail-mediated tight attachment to host cells presumably ensures that Yop delivery is rapid and efficient114. Aside from its adhesive properties, Ail also confers resistance to serum killing115 in all three human pathogenic yersiniae. It is apparent that Ail plays a more prominent role in the virulence of Y. pestis, which is presumably owing to the fact that the other prominent virulence factors contributing many of the Ail functions in Y. pseudotuberculosis and Y. enterocolitica are dysfunctional.\n\n\nPsa – the pH6 antigen\n\nThe chromosomally encoded pH6 antigen (Psa) was originally identified in Y. pestis as a surface antigen expressed at mammalian body temperatures at pH values similar to those found in phagolysosomes116. It was subsequently found to cause the agglutination of erythrocytes113,117. Further investigation revealed a cell surface complex composed of aggregates of a 15 kDa protein (PsaA) that requires two regulators, PsaE and PsaF, for maximal induction118,119. PsaA possesses a flexible fimbrial structure that is highly expressed during macrophage infection120. Biochemical examination of Psa reveals that it binds to β1-linked galactosyl residues in glycosphingolipids121, mainly of the type found in apolipoprotein-B-containing lipoproteins in human plasma, such as low-density lipoprotein (LDL) and in lipid rafts in macrophage membranes122. Furthermore, Psa acts as a bacterial Fc receptor, binding human immunoglobulin (IgG) but not reacting with rabbit, mouse, or sheep IgG123. As with the other adhesins, the activities of Psa appear to mediate Yop secretion. Y. pseudotuberculosis and Y. enterocolitica both produce a surface protein analogous to Psa but it is referred to as MyfA. Both Psa and MyfA coat the bacterial surface with a fibrillar matrix120,124 and in Y. pseudotuberculosis MyfA promotes attachment to tissue culture cells and haemagglutination113.\n\n\nY. pestis plasmid-specific virulence factors\n\nApart from the T3SS virulence plasmid, two other plasmids, pPCP and pMT (sometimes referred to as pFra) that are unique to Y. pestis, possess additional virulence factors. pPCP encodes the plasminogen activator Pla protease/adhesin (Figure 3). Pla converts plasminogen to plasmin125,126, which then degrades extracellular matrices and confers on Y. pestis the ability to rapidly invade the host and migrate to lymphatic tissues127,128. The over-activation of plasmin results in laminin and fibrin clot degradation, exacerbating migration across host barriers129, which is further compounded by the activities of Pla as an adhesin and an invasin130,131. pMT is responsible for the production of a murine toxin that is required during the colonisation of fleas132.\n\n\nConcluding remarks\n\nOver the last three decades, a considerable amount of detailed knowledge has accumulated that has enabled us to understand how the yersiniae colonise tissues and combat host defences during infection. While the Yersinia T3SS is perhaps the best understood system of its kind, many questions remain unanswered. For example, fully elucidating the function of YopM will offer an important step change, as will understanding more clearly the global molecular mechanisms that underpin the regulatory relationships that must exist between the T3SS system and the adhesins. It is also important to try to understand the relationships that exist between the different adhesins, how they compensate for each other, and which environmental signals dictate their site-specific expression. Finally, although the structures of many of the adhesins have been elucidated, there is certainly a need to better understand how they interact with different host ligands. While significant progress has been made in defining this sophisticated and finely tuned arsenal of virulence determinants, much more work is required to fully appreciate the success of the yersiniae as pathogens.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAchtman M, Zurth K, Morelli G, et al.: Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A. 1999; 96(24): 14043–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAchtman M, Morelli G, Zhu P, et al.: Microevolution and history of the plague bacillus, Yersinia pestis. Proc Natl Acad Sci U S A. 2004; 101(51): 17837–42. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPrentice MB, Rahalison L: Plague. Lancet. 2007; 369(9568): 1196–207. PubMed Abstract | Publisher Full Text\n\nStenseth NC, Atshabar BB, Begon M, et al.: Plague: past, present, and future. PLoS Med. 2008; 5(1): e3. 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[
{
"id": "14362",
"date": "14 Jun 2016",
"name": "James Bliska",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14363",
"date": "14 Jun 2016",
"name": "Andrew Roe",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
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https://f1000research.com/articles/5-1370
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https://f1000research.com/articles/5-1369/v1
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14 Jun 16
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{
"type": "Review",
"title": "Recent advances in understanding apicomplexan parasites",
"authors": [
"Frank Seeber",
"Svenja Steinfelder",
"Svenja Steinfelder"
],
"abstract": "Intracellular single-celled parasites belonging to the large phylum Apicomplexa are amongst the most prevalent and morbidity-causing pathogens worldwide. In this review, we highlight a few of the many recent advances in the field that helped to clarify some important aspects of their fascinating biology and interaction with their hosts. Plasmodium falciparum causes malaria, and thus the recent emergence of resistance against the currently used drug combinations based on artemisinin has been of major interest for the scientific community. It resulted in great advances in understanding the resistance mechanisms that can hopefully be translated into altered future drug regimens. Apicomplexa are also experts in host cell manipulation and immune evasion. Toxoplasma gondii and Theileria sp., besides Plasmodium sp., are species that secrete effector molecules into the host cell to reach this aim. The underlying molecular mechanisms for how these proteins are trafficked to the host cytosol (T. gondii and Plasmodium) and how a secreted protein can immortalize the host cell (Theileria sp.) have been illuminated recently. Moreover, how such secreted proteins affect the host innate immune responses against T. gondii and the liver stages of Plasmodium has also been unraveled at the genetic and molecular level, leading to unexpected insights. Methodological advances in metabolomics and molecular biology have been instrumental to solving some fundamental puzzles of mitochondrial carbon metabolism in Apicomplexa. Also, for the first time, the generation of stably transfected Cryptosporidium parasites was achieved, which opens up a wide variety of experimental possibilities for this understudied, important apicomplexan pathogen.",
"keywords": [
"Apicomplexa",
"parasites",
"Plasmodium",
"T. gondii",
"CRISPR/Cas9"
],
"content": "Introduction\n\nApicomplexa comprise a large phylum of single-celled, obligate intracellular protozoan organisms that all have a parasitic lifestyle. Among the more than 6000 named and probably more than one million unnamed species1 are some of great public health and economic relevance, since they cause severe diseases in humans and livestock, affecting millions each year2–6. Therefore, increased knowledge about their biology in general, e.g. to exploit vulnerabilities, and their interaction with the host organism, e.g. to stimulate the immune system, is of great importance and promises major benefits in understanding and combating the diseases they cause. In addition, they possess a fascinating biology as intracellular eukaryotes thriving within another eukaryotic cell (Figure 1), which clearly sets them apart from other pathogens like bacteria and viruses. Taken together, these are all good reasons to pay attention to these fascinating organisms. This review will focus on four important genera.\n\nTransmission electron micrograph of the intracellular Toxoplasma gondii tachyzoite stage as one example of Apicomplexa. Shown are parasites inside an infected fibroblast host cell, residing within a parasitophorous vacuole (PV), which is surrounded by a membrane (parasitophorous vacuolar membrane, PVM, in blue) (left). On the right, some host and parasite organelles are outlined. The apicoplast is a plastid-derived organelle harboring some essential metabolic pathways. Micronemes are specialized secretory organelles at the apical end that store proteins important for gliding motility and host cell invasion130. The role of dense granules and rhoptries are detailed in the text. This basic organellar setup is shared with Plasmodium sp.\n\nPlasmodium falciparum and four other Plasmodium species that affect humans cause malaria, a mosquito-transmitted, potentially deadly disease. According to the latest data from the World Health Organization (WHO), the number of deaths due to malaria has declined by 48% between 2000 and 2015 but the disease still causes the loss of ca. 438,000 lives each year of the 218 million people infected, mostly children under 5 years of age5.\n\nAn estimated one-third of the human population is chronically infected with Toxoplasma gondii leading to toxoplasmosis. It can cause severe symptoms in newborns (e.g. encephalitis and ocular disease) when a previously non-infected mother contracts the infection during pregnancy by ingesting infectious stages of T. gondii via contaminated food, water, or dust7. The latest numbers from WHO rank toxoplasmosis highest with respect to the overall lifelong disease burden among those foodborne diseases caused by protozoan parasites8.\n\nThe same study identified cryptosporidiosis (caused by two Cryptosporidium species, Cryptosporidium hominis and Cryptosporidium parvum) as the second most important disease in this class. Worldwide, each of these species infects 8–10 million people per year, but in addition they also cause considerable disease in livestock.\n\nLastly, Theileria parva, a tick-transmitted apicomplexan of ruminants, leads to the death of more than 1 million cattle per year in sub-Saharan Africa, causing costs of >300 million US$. This has severe socio-economic consequences for those regions4.\n\nT. gondii, and to a lesser extent Plasmodium sp., has also gained increased attention from cell biologists owing to a number of unique mechanisms of host cell entry, division, motility, etc., thereby becoming model organisms for such aspects9. Both medical importance and model character are reflected by the overall citations in PubMed (several thousands from January 2013–December 2015 for the four taxa), making it impossible to cover even the most interesting discoveries of the last 2–3 years in all areas of apicomplexan research in reasonable depth. Consequently, only a few, admittedly subjective highlights in the fields of cell and molecular biology, biochemistry, and immunology as well as aspects concerning drugs will be mentioned here. These were selected because they are expected to make a major impact in the following years or offer explanations for puzzling unexplained observations in apicomplexan biology.\n\n\nMethodological advances as game-changers\n\nMany new findings require new technologies at first, and with the advent of next-generation sequencing (NGS) techniques10, the number and quality of apicomplexan genome sequences and respective transcriptomes have been growing considerably, allowing insights and discoveries that would have been hard or impossible to gain a few years ago11–13. The genetic tools are most advanced for T. gondii and Plasmodium, and further specifics can be found in recent articles14–17. Moreover, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system18 and related techniques started a wave in 201219,20 that swept over the life sciences and since then has also reached the Apicomplexa. It has allowed targeted deletions, mutations, or gene additions so far in Plasmodium, T. gondii, and Cryptosporidium with unprecedented ease21–25, as will be illustrated here for the latter.\n\nCryptosporidium sp. have some unique biological aspects within the phylum, which are of great evolutionary interest, like the presence of only a rudimentary mitochondrion (mitosome) or the absence of the secondary plastid called apicoplast26,27. However, the lack of protocols for efficient long-term propagation in cell culture, reverse genetics, and thus methods to mark the parasites with fluorescent proteins has long precluded a broader analysis of many of those aspects. Application of CRISPR/Cas9, together with a number of smart optimizations of established techniques, plus the transfer of transfected parasites directly into the intestine of immune-deficient mice and their subsequent selection therein, changed the game22. This allowed for the first time the establishment of stably transfected C. parvum parasites and opens up a whole variety of options. For example, parasites expressing a reporter enzyme like luciferase will enable comprehensive drug screening efforts, allowing the tackling of an urgent need. Likewise, generation of genetically attenuated parasites by multi-gene deletions can be envisaged as a means to develop oral vaccines for livestock (or even humans). Effective vaccines for the former would minimize the contamination of the environment with excreted infectious oocysts. Finally, many fascinating cell biological aspects can now be followed and analyzed using fluorescent sporozoites.\n\n\nAll about artemisinin in a nutshell: biotechnological production, mode of action, and the emergence and nature of Plasmodium drug resistance\n\nIn 2015, the Nobel Prize in Physiology or Medicine was in part awarded to Chinese scientist Youyou Tu for her major contributions to the discovery of artemisinin in the 1970s28,29. It is the ingredient of a traditional Chinese herbal anti-malarial treatment that, when metabolized in situ to dihydroartemisinin, very efficiently kills Plasmodium sp.. Artemisinin combination therapies (ACTs) are currently the drugs of choice and recommended by WHO for treating P. falciparum infections worldwide, largely because drug resistance against other available compounds precludes their further use in many areas of Africa and South-East Asia (SEA)30. ACTs consist of fast-acting artemisinin (or its derivatives) and less-potent, long-acting partner drugs. Artemisinin’s mode of action is unique in the sense that not a single protein or cell component but a multitude of parasite molecules are targeted by the compound via the generation of highly reactive endoperoxide-derived radicals31. These affect at least 124 parasites but few, if any, host proteins (at the level of currently applied experimental resolution), as a recent study reported32. This probably leads to the observed cellular stress response and increased molecular tagging of the affected proteins for disposal by the “cellular garbage can”, the proteasome33. Many of these proteins are known or suspected to be essential for parasite growth, and their concerted damage by an unselective mechanism like oxidative damage could be expected to make it fairly difficult for the parasite to develop resistance (see below).\n\nOnly one plant is known to produce artemisinin, Artemisia annua L., and extraction yields do not exceed 0.5%. Therefore, the molecular deciphering of its biosynthesis34 and the subsequent biotechnological production of artemisinic acid, the key precursor from which artemisinin and other derivatives can be derived by straightforward chemical synthesis, was a major breakthrough35,36. The genes required for this pathway were engineered into a yeast strain that can now produce artemisinic acid with a yield of 25 g/L of fermentation broth36. Notably, although developed by a company, all intellectual property rights have been provided free of charge.\n\nUnfortunately, this success story has recently been dampened by the emergence (again!) of resistance phenotypes in SEA37,38. Resistance is defined as a parasite clearance half-life of at least 5 hours following ACT treatment, whereas non-resistant Plasmodium parasites are all killed earlier. The problem lies in the fact that delayed clearance (parasites are still killed, but slower) exposes the surviving organisms to the second drug in ACT, increasing the chance that resistance to this partner compound develops. Clearly, understanding the mechanism of artemisinin resistance is of utmost importance to be able to counteract and change drug regimens or composition.\n\nHere, NGS and related techniques come into play. Sequencing resistant P. falciparum isolates directly from patients, a number of recent studies provided solid evidence for multiple mutations in a gene called kelch13 (K13), which are associated with increased resistance39–42. Transcriptomics identified an additional protein presumably involved, phosphatidylinositol-3 kinase (PI3K)43 (for details, see Figure 2).\n\nIn wild-type (wt) parasites, binding of K13 (blue, based on 10.2210/pdb4yy8/pdb) to an as-yet-unknown transcription factor (TF, red) usually leads to the tagging of TF with ubiquitin, followed by its subsequent degradation via the proteasome (a). This regulatory mechanism is abolished when mutated K13 is no longer able to bind efficiently to TF, thereby preventing ubiquitin tagging and subsequent proteasomal degradation (b). This results in increased transcription of genes and production of the corresponding proteins involved in antioxidant defense. Their activity allows counteracting the oxidative damage brought upon them by artemisinin. In an alternative model43, the low phosphatidylinositol-3-phosphate (PI3P) levels usually found in artemisinin-sensitive parasites is maintained by K13 binding to its kinase phosphatidylinositol-3-kinase (PI3PK, blue), its ubiquitination (similar to TF) and subsequent degradation (a). Loss of this regulation leads to increased PI3PK levels, followed by a buildup of PI3P (c). Higher levels are presumably responsible for parasite growth in the schizont stage via promoting membrane biogenesis and fusion events during parasite growth. In addition, artemisinin combination therapy (ACT) is able to directly inhibit PI3PK. Both mechanisms could also benefit from the observed slowed-down growth and upregulation of the unfolded protein response pathway44, giving treated parasites more time to repair damaged proteins before they progress through the rest of their lifecycle in red blood cells. Figure adapted from 37.\n\nAnother transcriptomics study of 1000 (!) clinical P. falciparum isolates provided evidence that a population of parasites exists that is slowed down in growth and shows an upregulated so-called “unfolded protein response” pathway44. This might allow parasites to repair proteins that were oxidatively damaged by artemisinin’s mode of action (see above) and progress through the rest of their lifecycle in red blood cells. The reduced growth would give them more time to do so. Puzzlingly, this report found no association between PI3K transcript levels and either parasite clearance half-life or K13 mutations. Evidently, more efforts are required to reconcile all the observations and to fully understand resistance development. Nevertheless, in a very short time, from the initial observation of delayed clearance times of ACT in SEA to the current studies, immense progress has been made in understanding the obviously very complex artemisinin resistance mechanism(s). Technological breakthroughs were just there at the right time to face this challenge.\n\n\nMitochondrial metabolism – similar but still significantly different\n\nCondensing parasitism to the meaning of the Greek word παράσιτος (parásitos; person who eats at someone else's table), and regarding biochemistry as the underlying science to reveal this eating behavior, there is an obvious and long-standing interest in understanding apicomplexan metabolism, not least because enzymes can make good drug targets45. Again, technological advances in the field of metabolomics, together with gene knockouts, have greatly helped finding some answers to who eats what, when, and how.\n\nMitochondrial carbon metabolism in Apicomplexa is central to the generation of energy and several precursors of other pathways that occur outside the organelle, like pyrimidine and heme biosynthesis46–49. One fundamental energy-generating system in most eukaryotes is the tricarboxylic acid (TCA) cycle in this organelle. It leads to complete oxidation of carbohydrates, lipids, and amino acids, thus allowing much greater ATP generation through the electron transport chain (ETC) than e.g. breakdown of glucose via glycolysis. T. gondii and Plasmodium sp. can get along with an energy supply derived only from glycolysis as long as they are living in glucose-rich environments. In fact, most (but not all) TCA cycle enzymes in P. falciparum could be knocked out with only little influence on the blood stage forms50. Nevertheless, for a number of reasons, it seemed that a TCA cycle and ETC were operating in both organisms. For instance, several recent studies50–55 have shown that the development of Plasmodium berghei (a rodent model for P. falciparum) in the mosquito is not possible without a functional TCA cycle or ETC (summarized in 47). These studies have highlighted the great flexibility of both Plasmodium sp. and T. gondii with regard to substrate utilization and adaptation of carbon metabolism to different host environments.\n\nHowever, an unresolved mystery was the experimentally well-proven absence of a key enzyme complex, pyruvate dehydrogenase (PDH), in the mitochondrion of Apicomplexa that would allow these organisms to feed glucose-derived pyruvate into the TCA cycle47,48 (Figure 3a). This longstanding conundrum has now been solved, and it could be shown that a structurally related mitochondrial enzyme complex, branched-chain alpha-ketoacid dehydrogenase (BCKDH) usually involved in the degradation of branched-chain amino acids (BCAAs), has evolved to also take over the PDH task, generating acetyl-CoA from pyruvate51 (Figure 3a). Since pyruvate and the usual substrates for BCKDH are structurally quite similar (Figure 3b), it is likely that only few (so far unknown) mutations were necessary to acquire the required substrate specificity. It is an illuminating example that holes in metabolic pathways predicted from genome data are not necessarily an annotation problem but can reflect evolutionary processes of reductive evolution46,48.\n\na) Current model of how the tricarboxylic acid (TCA) cycle is fed with substrates in both Toxoplasma gondii (blue) and Plasmodium berghei (red; in common with T. gondii) and converted to acetyl-CoA. The latter then enters the TCA cycle. BCAA degradation in T. gondii can lead to toxic propionyl-CoA, which could be detoxified by the 2-methylcitrate (MC) cycle. Its physiological importance for the different T. gondii stages is currently ill defined, as both BCAA degradation and MC cycle are dispensable in tachyzoites131. Abbreviations: α-KG, α-ketoglutarate; BCAT, branched-chain amino acid transferase; BCKDH, branched-chain keto acid dehydrogenase; Fum, fumarate; Cit, citrate; iCit, isocitrate; Mal, malate; OAA, oxaloacetic acid; PEP, phosphoenolpyruvate; Suc, succinate. Figure adapted and redrawn from 51. b) Structural similarities of substrates for pyruvate dehydrogenase (PDH) and branched-chain keto acid dehydrogenase (BCKDH), respectively. The framed reaction scheme illustrates the overall generation of CoA compounds and CO2 from the substrates catalyzed by the two enzymes.\n\nReductive evolution aims to reduce the considerable metabolic burden of gene and protein synthesis when the function of lost genes can be fulfilled otherwise56,57. Importantly, in this case, parasitism wasn’t the driving force, since free-living dinoflagellates that share a common ancestor with Apicomplexa are also devoid of a mitochondrial PDH but possess BCKDH46. Interestingly, while Plasmodium sp. have lost the entire BCAA degradation pathway of enzymes operating before and after BCKDH46,48, thus saving even more on gene and protein synthesis, T. gondii as well as non-parasitic photosynthetic algae related to Apicomplexa have kept it58.\n\n\nUnderstanding apicomplexan host cell manipulation – it all depends on protein export\n\nOne of the most fascinating aspects of the biology of Apicomplexa in general is their remarkable capacity to manipulate their respective host cells to suit their own demands. This causes changes in host cell signaling, nutrient delivery to the parasite, and evasion of host immunity59–62. Amongst the many strategies leading to this outcome, unique examples are provided by T. parva and Theileria annulata.\n\nBoth species have the ability to immortalize (transform) their host cells, which resembles in many aspects cancerous cell transformation. T. parva transforms bovine B and T lymphocytes, whereas T. annulata, besides B cells, also transforms macrophages and dendritic cells63. Activation of the c-Jun pathway, a signaling cascade involved in controlling many cellular processes including proliferation64, has been shown previously to be required for Theileria-induced transformation65,66. However, how this is accomplished at the molecular level has long been elusive.\n\nNow, Marsolier et al.67 have reported the identification of a parasite-encoded enzyme called peptidyl-prolyl isomerase (PPIase), a homologue of human parvulin (hPIN1), as the transforming agent. Upon export into the host cell, the Theileria PPIase, like the human homolog, binds to a host protein (an ubiquitin ligase that controls the levels of c-Jun), leading to its destabilization and subsequent degradation. This in turn results in higher levels of c-Jun and eventually leads to cell transformation. These surprising findings go beyond parasite biology, as they also define prolyl-isomerization as a conserved mechanism that is important in cancer development.\n\nT. gondii, amongst other Apicomplexa, is also a known expert in manipulation of the host cell at various levels via the secretion upon host cell entry of effector proteins68,69, the number of which (currently around 8058) is still increasing70–72. The so-called rhoptry proteins (ROPs) are injected into the host cell cytosol during the actual invasion process via special organelles called rhoptries73. A number of so-called GRA proteins derive from other organelles, the dense granules, and are delivered into the host cytosol after the parasite has formed its vacuole wherein it resides74. Consequently, and in contrast to the ROPs, their mechanism of trafficking must include a way to pass this membrane structure. This aspect recently gained increased attention75.\n\nPrecedence for this transport pathway came from the one that had been described for erythrocytic stages of Plasmodium. There, one molecular entity in the vacuolar membrane named “Plasmodium translocon of exported proteins” (PTEX) was recently described, which transports many parasite proteins into the cytosol of the red blood cell76–79. Delivery to PTEX depends in turn on prior export of proteins out of the parasite cell into the vacuolar space, which requires cleavage by a specific protease (plasmepsin 5) at a particular sequence motif called “Plasmodium export element” (PEXEL)79. This PEXEL motif allows the identification of many but not all proteins that are to be exported into the host cytosol80.\n\nIt made sense to also look for such a motif in T. gondii – and it was found for numerous known GRA proteins75. Now, several groups recently reported the importance of export of T. gondii aspartyl protease ASP5 (the homolog to plasmepsin 5 in Plasmodium), which resides in the parasite’s Golgi and processes those GRA proteins containing the PEXEL-like motif70,72,81. However, not all GRAs that depend on ASP5 for export contain a PEXEL-like motif. In Plasmodium, plasmepsin 5 is an essential gene already under in vitro growth conditions. This fact allowed the development of a very potent inhibitor of this crucial enzyme and that was subsequently used for structural studies82. In contrast, T. gondii parasites with a deleted ASP5 grow fine in cell culture but are much less virulent in a mouse model than the highly virulent parental strain. The very same phenotype has been described for a knockout strain of the ASP5-processed GRA1483. Together, this indicates the crucial importance of a repertoire of exported proteins during a natural infection. One reason for this might be the observed reduced migration of infected dendritic cells (which are known to be misused by T. gondii as Trojan horses to reach the brain84), thereby lowering the dissemination within the host72. Interestingly, this disparate phenotype upon gene deletion of in vitro and in vivo growth, together with a severe impact on protein export into the host, is shared with another recently reported T. gondii parasitophorous vacuolar membrane (PVM)-resident protein, Myc regulation 1 (MYR1)85. Whether MYR1 is part of the T. gondii “translocon of exported proteins” needs to be determined, since e.g. Plasmodium sp. lack a homolog of MYR1. In addition, a T. gondii homolog of the putative PTEX pore protein EXP286, GRA17, is involved in the translocation of only small molecules through the PVM but apparently not of proteins87. This indicates that both species use very different molecular complexes for protein export into the host cell.\n\nAnother distinctive difference to Plasmodium sp. is that in the absence of ASP5, certain GRA proteins fail to be exported into the host’s cytosol and then further on into its nucleus. Here, they cause large disturbances in the transcriptome of the infected cell70,72. Plasmodium sporozoites, the stage that infects hepatocytes, apparently do not possess dense granules61. Nevertheless, large transcriptomic changes in liver cells occur upon infection88. Apparently, in both parasite species different effectors accomplish similar things, i.e. they modify their host cells to optimize their own survival therein.\n\nThe T. gondii studies further indicate that many more exported effector proteins besides the known ROPs and GRAs need to be identified to fully understand the ways this parasite manipulates its host cell. The ASP5 mutants will be a valuable source in this respect. More studies will be required to understand the details and evolution of this crucial mechanism in the Apicomplexa89.\n\n\nInnate immune defense always starts at the host-parasite interface\n\nInnate immune responses are the immediate answer to an infection. They are triggered by the recognition of pathogen-derived molecules via evolutionarily conserved host receptors90. In the last two decades, numerous studies found that innate sensing of apicomplexan infection is mediated by membrane-bound and cytoplasmic so-called pathogen recognition receptors (PRRs) such as Toll-like receptors (TLRs) and Nod-like receptors (NLRs)91,92. Early sensing of infection by these pathways in antigen-presenting cells bridges the innate and adaptive immune responses by licensing them to interact with naïve, antigen-specific CD4+ and CD8+ T cells to stimulate them to become effector cells capable of producing cytokines and/or cytotoxic molecules. Efficient control of T. gondii and Plasmodium sp. infections is mediated by T and NK cell-derived interferon-gamma (IFN-γ)93,94. This cytokine triggers the activation of very diverse effector pathways, e.g. NO production in infected host cells95 or induction of immunity-related GTPase (IRG) proteins (a family of rodent IFN-γ-induced GTPases) or GBP proteins (IFNγ-inducible guanylate-binding proteins) that damage the PVM (Figure 1), thereby mediating parasite death96–101. Recent studies emphasized the importance of GBP1100, GBP2101, and the cooperation of IRGs with GBPs99 in cell-autonomous immunity and anti-parasitic resistance.\n\nWhile the effects of IRGs on T. gondii in infected mice were known for some time, it became only recently apparent that laboratory mice and wild Mus musculus show enormous sequence diversity in particular genes of this family, with great and unexpected consequences for infection102. For decades, T. gondii strains were defined as being highly virulent in various strains of common laboratory mice because even a single parasite could kill them within days. Unexpectedly, those strains totally failed to reproduce this phenotype in wild mice. This is largely due to a highly polymorphic IRG allele that confers resistance against virulent parasites by interfering with their virulence factors of the ROP family of protein kinases102. The study is a striking example that lab mice and their wild counterparts can show very different responses to identical immunological challenges.\n\nProtective immunity against T. gondii is governed by an IL-12-triggered Th1-type immune response, which involves NK cells, CD4+ effector cells, and CD8+ cytotoxic T cells as sources of IFN-γ93. In order to induce its production by these cells, the innate arm of the immune system needs to sense the infection and relay this information into IL-12 production as an igniting factor for the ensuing Th1 response. Control of the infection is dependent on MYD88103, an adaptor molecule common to several TLRs, and the parasite-derived TLR-ligand T. gondii profilin (TgPRF). The latter binds TLR11, which triggers a signaling cascade that stimulates IL-12 production by dendritic cells59,104. However, TLR11 deficiency only modestly affects the survival of T. gondii-infected mice. Only recently was TLR12 shown to be involved in host resistance to T. gondii by recognizing TgPRF105 and cooperating with TLR11 to induce IL-12 in macrophages and dendritic cells106.\n\nOther studies demonstrate the involvement of additional pathogen-derived molecules and PRRs in starting an immune response by contributing to the stimulation of IL-12 production. A recent study employed mice carrying a mutation in UNC93B1, a molecule involved in subcellular trafficking of endosomal TLRs107. These mice are highly susceptible to experimental toxoplasmosis and their phenotype is not recapitulated by mice deficient in nucleic acid sensing (TLR3-/TLR7-/TLR9-deficient) but rather by mice deficient in TLR7/TLR9 and TLR11, highlighting the redundancy of pathogen recognition in those animals108 (Figure 4a). Humans lack TLR12 and harbor TLR11 as a pseudogene but produce high levels of pro-inflammatory cytokines in response to live phagocytosed parasites and parasite RNA and DNA. Thus, nucleic-acid-sensing TLRs seem to be the PRR operating in both hosts108,109, while recognition of TgPRF by the TLR11/12 pathway is most likely an adaptation of rodents being a major intermediate host for T. gondii109.\n\na) Simplified model of how dendritic cells sense T. gondii infection. Toll-like receptor 11 (TLR11) and TLR12 are the major receptors for the T. gondii-derived protein profilin (TgPFR). Interferon regulatory factor 8 (IRF8)+ dendritic cells (DCs), in particular CD8+ DCs, have a crucial role in detecting T. gondii profilin and in the subsequent induction of interleukin (IL)-12 production downstream of myeloid differentiation primary-response protein 88 (MYD88). A novel MYD88-dependent signaling pathway that depends on activation of IRF8 is an explanation for the potent induction of IL-12 expression by CD8α+ DCs that have been exposed to T. gondii, but the direct connection between MYD88 and IRF8 has not been established. In addition, both TLR7 and TLR9 have been implicated in detecting T. gondii RNA and genomic DNA, respectively. UNC93B1 (UNC), an endoplasmic reticulum-resident protein, has a central role in the function of all of the depicted endosomal TLRs and is essential for resistance to T. gondii (modified and redrawn from 132). b) Schematic representation of the proposed sequence of events occurring in liver-resident cells upon Plasmodium sp. infection. Plasmodium RNA triggers a type I interferon response via activation of the cytoplasmic RNA sensor Mda5 and other unknown receptors, mitochondrial antiviral signaling protein (Mavs), and the transcription factors interferon regulatory factor 3 (Irf3) and Irf7 (modified and redrawn from 111).\n\nIn contrast to T. gondii, which can infect virtually any nucleated cell, Plasmodium sp. are restricted to infecting hepatocytes and erythrocytes. Upon injection of Plasmodium sporozoites by the mosquito vector into the skin of the host, they first migrate to the liver and invade hepatocytes where they massively replicate. Early work on innate sensing of Plasmodium revealed that hemozoin-containing parasite DNA is recognized by TLR9 and mediates proinflammatory cytokine production by dendritic cells110, but little was known about the hepatic immune response. Strikingly, and in stark contrast to the erythrocytic cycle, which causes the well-known malaria symptoms like recurrent fever, this initial contact between the parasite and its host cell is without clinical signs. This could be taken as evidence for an immunologically silent hepatic stage of the infection. However, recently it was shown that hepatocytes do respond to P. berghei sporozoite invasion with induction of a type I IFN response. Parasitemia in liver and red blood cells was increased and leucocyte recruitment was decreased in type I IFN receptor-deficient (Ifnar-/-) mice, highlighting the important role of type I IFN at this early phase of infection111. The authors proposed a mechanism involving sensing of Plasmodium RNA by the cytoplasmic RNA receptor Mda5 and signaling via the mitochondrial antiviral signaling protein (Mavs) and the transcription factors Irf3 and Irf7, finally leading to transcription of IFN-α and IFN-β (Figure 4b). A potential link among IFN-α, cell recruitment, and parasite elimination could be IFN-γ-secreting NK T cells, suggested by a recent study using the related species Plasmodium yoelii112. However, a successful immune response often comes at a price, since a type I IFN response also causes malaria-associated immunopathology, such as liver damage and brain pathology113,114.\n\n\nOutlook\n\nObviously, we could give only a subjective glimpse of the recent exciting developments in understanding apicomplexan biology. What will come next?\n\nOne can probably barely overestimate the impact that CRISPR/Cas9 technology will have on Apicomplexa research. It is expected that genome-wide CRISPR/Cas9 gene knockouts, similar to what has been described for mammals115, will also be published soon for Apicomplexa (preliminary data have been reported already for T. gondii116). This will allow researchers to qualify genes as being either essential or not under different in situ conditions and to discover so far unknown phenotypes. For instance, parasites deficient in molecules known to target the innate immune system (but also knockdown of host PRRs, especially in primary human cells) will reveal pathways that are necessary for innate sensing and hence parasite control in rodents and humans and help to answer the question of whether actual infection, phagocytosis, or mere contact (“kiss and spit”) is required for CD4+ and CD8+ T cell responses.\n\nMoreover, microscopy-based genome-wide screens on CRISPR/Cas9-generated knockouts or fluorescently tagged proteins can be envisaged to follow117. It would allow testing for function and localization of proteins that could not be achieved with this precision, speed, and coverage so far.\n\nIn particular, the less-studied stages (e.g. sexual stages within the mosquito [Plasmodium] or the cat [T. gondii]) will be of scientific interest. For the latter, establishing feline organoid cultures that would allow in vitro culture of the sexual stages will be crucial118. One of the most eminent questions in this respect is why does sex only take place in cats when this parasite is otherwise so extremely promiscuous in its host range? Together with the advent of modern metabolomics and gene knockouts, the answer to this and other questions might be within reach in the next few years.\n\nSince Apicomplexa are pathogens, the development of new drugs and drug target candidates will of course remain a major driver for studying these parasites, and it is inevitable that there will be a big gain in understanding of the underlying molecular processes. Recent studies on crucial metabolic pathways for lipids119,120, sugars121, and isoprenoids122 have shown more potential metabolic vulnerabilities of apicomplexan parasites. Moreover, a new class of highly active anti-malarial compounds has already been described123,124, and one of them, the spiroindolone KAE609, has shown very promising results in recent phase II clinical trials against uncomplicated malaria125–127. Although drug resistance in the laboratory has already been described for spiroindolones, knowledge about its reported mechanism123,128,129 will hopefully help in designing strategies to pre-emptively delay the spread of resistance in nature once KAE609 has been brought onto the market. However, the race is bound to start all over again – winner currently unknown.\n\n\nAbbreviations\n\nACTs, artemisinin combination therapies; BCAA, branched-chain amino acid; ASP5, aspartyl protease 5; BCAT, branched-chain amino acid transferase; BCKDH, branched-chain alpha-ketoacid dehydrogenase; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; Cas, CRISPR associated; ETC, electron transport chain; hPINI, human parvulin; IFN-γ, interferon-gamma γ; IFNα/β, interferon α/β; IRG, immunity related GTPases; K13, kelch13; Mavs, mitochondrial antiviral signaling protein; MC, 2-methylcitrate; MYR1, Myc regulation 1; NGS, next-generation sequencing; NOD, Nod-like receptor; PDH, pyruvate dehydrogenase; PEXEL, Plasmodium export element; PI3K, phosphatidylinositol-3-kinase; PI3P, phosphatidylinositol-3-phosphate; PPIase, peptidyl-prolyl isomerase; PRR, pathogen recognition receptor; PTEX, Plasmodium translocon of exported proteins; PVM, parasitophorous vacuolar membrane; ROPs, rhoptry proteins; SEA, South-East Asia; TCA, tricarboxylic acid; TgPFR, Toxoplasma gondii-derived protein profilin; TLR, Toll-like receptor.\n\nDedicated to the memory of Klaus Lingelbach, a devoted Plasmodium scientist and a mentor of Frank Seeber.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe authors are senior members of the Research Training Group 2046 (GRK 2046) \"Parasite Infections: From Experimental Models to Natural Systems\" funded by the Deutsche Forschungsgemeinschaft (DFG).\n\n\nAcknowledgements\n\nWe apologize to all whose studies were not mentioned owing to space constraints or were missed owing to our ignorance. We thank Michael Laue and Florian Müller for providing the image for Figure 1 and Toni Aebischer and Totta Kasemo for comments on the manuscript.\n\nDedicated to the memory of Klaus Lingelbach, a devoted Plasmodium scientist and a mentor of Frank Seeber.\n\n\nReferences\n\nAdl SM, Leander BS, Simpson AG, et al.: Diversity, nomenclature, and taxonomy of protists. Syst Biol. 2007; 56(4): 684–9. 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PubMed Abstract | Publisher Full Text\n\nRocha BC, Marques PE, Leoratti FM, et al.: Type I interferon transcriptional signature in neutrophils and low-density granulocytes are associated with tissue damage in malaria. Cell Rep. 2015; 13(12): 2829–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEdwards CL, Best SE, Gun SY, et al.: Spatiotemporal requirements for IRF7 in mediating type I IFN-dependent susceptibility to blood-stage Plasmodium infection. Eur J Immunol. 2015; 45(1): 130–41. PubMed Abstract | Publisher Full Text\n\nShalem O, Sanjana NE, Zhang F: High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet. 2015; 16(5): 299–311. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSidik SM, Huet D, Lourido S: Genome-scale screens for Toxoplasma gene function using CRISPR/Cas9. In: Toxo13 - 13th international congress on Toxoplasmosis and Toxoplasma gondii research: 17-21 June 2015; Gettysburg, PA, USA. 2015; 49. Reference Source\n\nBoutros M, Heigwer F, Laufer C: Microscopy-based high-content screening. Cell. 2015; 163(6): 1314–25. PubMed Abstract | Publisher Full Text\n\nKlotz C, Aebischer T, Seeber F: Stem cell-derived cell cultures and organoids for protozoan parasite propagation and studying host-parasite interaction. Int J Med Microbiol. 2012; 302(4–5): 203–9. PubMed Abstract | Publisher Full Text\n\nGulati S, Ekland EH, Ruggles KV, et al.: Profiling the essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum. Cell Host Microbe. 2015; 18(3): 371–81. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nArroyo-Olarte RD, Brouwers JF, Kuchipudi A, et al.: Phosphatidylthreonine and lipid-mediated control of parasite virulence. PLoS Biol. 2015; 13(11): e1002288. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlume M, Nitzsche R, Sternberg U, et al.: A Toxoplasma gondii gluconeogenic enzyme contributes to robust central carbon metabolism and is essential for replication and virulence. Cell Host Microbe. 2015; 18(2): 210–20. PubMed Abstract | Publisher Full Text\n\nGuggisberg AM, Park J, Edwards RL, et al.: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites. Nat Commun. 2014; 5: 4467. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nVaidya AB, Morrisey JM, Zhang Z, et al.: Pyrazoleamide compounds are potent antimalarials that target Na+ homeostasis in intraerythrocytic Plasmodium falciparum. Nat Commun. 2014; 5: 5521. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRottmann M, McNamara C, Yeung BK, et al.: Spiroindolones, a potent compound class for the treatment of malaria. Science. 2010; 329(5996): 1175–80. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDiagana TT: Supporting malaria elimination with 21st century antimalarial agent drug discovery. Drug Discov Today. 2015; 20(10): 1265–70. PubMed Abstract | Publisher Full Text\n\nLehane AM, Ridgway MC, Baker E, et al.: Diverse chemotypes disrupt ion homeostasis in the Malaria parasite. Mol Microbiol. 2014; 94(2): 327–39. PubMed Abstract | Publisher Full Text\n\nWhite NJ, Pukrittayakamee S, Phyo AP, et al.: Spiroindolone KAE609 for falciparum and vivax malaria. N Engl J Med. 2014; 371(5): 403–10. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSpillman NJ, Allen RJ, McNamara CW, et al.: Na+ regulation in the malaria parasite Plasmodium falciparum involves the cation ATPase PfATP4 and is a target of the spiroindolone antimalarials. Cell Host Microbe. 2013; 13(2): 227–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpillman NJ, Kirk K: The malaria parasite cation ATPase PfATP4 and its role in the mechanism of action of a new arsenal of antimalarial drugs. Int J Parasitol Drugs Drug Resist. 2015; 5(3): 149–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSharma P, Chitnis CE: Key molecular events during host cell invasion by Apicomplexan pathogens. Curr Opin Microbiol. 2013; 16(4): 432–7. PubMed Abstract | Publisher Full Text\n\nLimenitakis J, Oppenheim RD, Creek DJ, et al.: The 2-methylcitrate cycle is implicated in the detoxification of propionate in Toxoplasma gondii. Mol Microbiol. 2013; 87(4): 894–908. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYarovinsky F: Innate immunity to Toxoplasma gondii infection. Nat Rev Immunol. 2014; 14(2): 109–21. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "14360",
"date": "14 Jun 2016",
"name": "Jeroen Saeij",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14361",
"date": "14 Jun 2016",
"name": "Julius Lukes",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1369
|
https://f1000research.com/articles/5-151/v1
|
09 Feb 16
|
{
"type": "Research Article",
"title": "Associations between chlorophyll a and various microcystin-LR health advisory concentrations",
"authors": [
"Jeffrey W. Hollister",
"Betty J. Kreakie",
"Betty J. Kreakie"
],
"abstract": "Cyanobacteria harmful algal blooms (cHABs) are associated with a wide range of adverse health effects that stem mostly from the presence of cyanotoxins. To help protect against these impacts, several health advisory levels have been set for some toxins. In particular, one of the more common toxins, microcystin-LR, has several advisory levels set for drinking water and recreational use. However, compared to other water quality measures, field measurements of microcystin-LR are not commonly available due to cost and advanced understanding required to interpret results. Addressing these issues will take time and resources. Thus, there is utility in finding indicators of microcystin-LR that are already widely available, can be estimated quickly and in situ, and used as a first defense against high concentrations of microcystin-LR. Chlorophyll a is commonly measured, can be estimated in situ, and has been shown to be positively associated with microcystin-LR. In this paper, we use this association to provide estimates of chlorophyll a concentrations that are indicative of a higher probability of exceeding select health advisory concentrations for microcystin-LR. Using the 2007 National Lakes Assessment and a conditional probability approach, we identify chlorophyll a concentrations that are more likely than not to be associated with an exceedance of a microcystin-LR health advisory level. We look at the recent US EPA health advisories for drinking water as well as the World Health Organization levels for drinking water and recreational use and identify a range of chlorophyll a thresholds. A 50% chance of exceeding one of the microcystin-LR advisory concentrations of 0.3, 1, 1.6, and 2 g/L is associated with chlorophyll a concentration thresholds of 23.4, 67.0, 83.5, and 105.8, respectively. When managing for these various microcystin-LR levels, exceeding these reported chlorophyll a concentrations should be a trigger for further testing and possible management action.",
"keywords": [
"Harmful Algal Blooms",
"Cyanotoxins",
"National Lakes Assessment",
"Conditional Probability Analysis",
"Cyanobacteria"
],
"content": "Introduction\n\nOver the last decade, numerous events and legislative activities have raised the public awareness of harmful algal blooms1–3. In response the US Environmental Protection Agency (US EPA) has recently released suggested microcystin-LR (one of the more common toxins) concentrations that would trigger health advisories4–6. Additionally, the World Health Organization (WHO) has proposed microcystin advisory levels for drinking water and a range of recreational risk levels7,8. While these levels and associated advisories are likely to help mitigate the impacts from harmful algal blooms, they are not without complications.\n\nOne of these complications is that they rely on available measurements of microcystin-LR. While laboratory testing (e.g., chromatography) remains the gold standard for quantifying microcystin-LR concentrations in water samples, several field test kits have been developed. Even though field tests provide a much needed means for rapid assessment, they are not yet widely used and are moderately expensive (approximately $150–$200 depending on the specific kit) with a limited shelf life (typically one year)9,10. Additionally, each technique requires nuanced understanding of the detection method (e.g., limit of detection, specific microcystin variants being measured, and sampling protocol).\n\nFortunately, microcystin-LR has been shown to be associated with several, more commonly measured and well understood components of water quality that are readily assessed in the field. For instance, there are small or hand held fluorometers that measure chlorohpyll a. Additionally, chlorophyll a is a very commonly measured component of water quality that is also known to be positively associated with microcystin-LR concentrations11,12. Yuan et al. (2014) explore these associations in detail and control for other related variables12. In their analysis they find that total nitrogen and chlorophyll a show the strongest association with microcystin. Furthermore, they identify chlorophyll a and total nitrogen concentrations that are associated with exceeding 1 μg/L of microcystin. Given these facts, it should be possible to identify chlorophyll a concentrations that would be associated with the new US EPA microcystin-LR health advisory levels for drinking water. Identifying these associations would provide another tool for water resource managers to help manage the threat to public health posed by cHABs and would be especially useful in the absence of measured microcystin-LR concentrations.\n\nIn this paper we build on past efforts and utilize the National Lakes Assessment (NLA) data and identify chlorophyll a concentrations that are associated with higher probabilities of exceeding several microcystin-LR health advisory concentrations6,8,13. We add to past studies by exploring associations with newly announced advisory levels and by also applying a different method, conditional probability analysis. Utilizing different methods strengthens the evidence for suggested chlorophyll a levels that are associated with increased risk of exceeding the health advisory levels as those levels are not predicated on a single analytical method. So that others may repeat or adjust this analysis, the data, code, and this manuscript are freely available via https://github.com/USEPA/microcystinchla.\n\n\nMethods\n\nWe used the 2007 NLA chlorophyll a and microcystin-LR concentration data13. These data represent a snapshot of water quality from the summer of 2007 for the conterminous United States and were collected as part of an ongoing probabilistic monitoring program13. Data on chlorophyll a and microcystin-LR concentrations are available for 1028 lakes. These data are available for download from the National Lake Assessment Data Site.\n\nWe used a conditional probability analysis (CPA) approach to explore associations between chlorophyll a concentrations and World Health Organization (WHO) and US EPA microcystin-LR health advisory levels14. Many health advisory levels have been suggested (Table 1), but lakes with higher microcystin-LR concentrations in the NLA were rare. Only 1.16% of lakes sampled had a concentration greater than 10 μg/L. Thus, for this analysis we focused on the microcystin concentrations that are better represented in the NLA data. These were the US EPA children’s drinking water advisory level of 0.3 μg/L (US EPA child), the WHO drinking water advisory level of 1 μg/L (WHO drinking), the US EPA adult drinking water advisory level of 1.6 μg/L (US EPA adult), and the WHO recreational, low probability of effect advisory level of 2 μg/L (WHO recreational)6–8.\n\nConditional probability analysis provides information about the probability of observing one event given another event has also occurred. For this analysis, we used CPA to examine how the conditional probability of exceeding one of the health advisories changes as chlorophyll a increases in a lake. We expect to find higher chlorophyll a concentrations to be associated with higher probabilities of exceeding the microcystin-LR health advisory levels. We also calculated 95% confidence intervals (CI) using 1000 bootstrapped samples. Thus, to identify chlorophyll a concentrations of concern we identify the value of the upper 95% CI across a range of conditional probabilities of exceeding each health advisory level. Using the upper confidence limit to identify a threshold is justified as it ensures that a given threshold is unlikely to miss a microcystin exceedance.\n\nAs both microcystin-LR and chlorophyll a values were highly skewed right, a log base 10 transformation was used. Additional details of the specific implementation are available at https://github.com/USEPA/microcystinchla. A more detailed discussion of CPA is beyond the scope of this paper, but see Paul et al.15 and Hollister et al.16 for greater detail. All analyses were conducted using R version 3.2.2 and code and data from this analysis are freely available as an R package at https://github.com/USEPA/microcystinchla.\n\nLastly, we assess the ability of these chlorophyll a thresholds to predict microcystin exceedance. We use error matrices and calculate total accuracy as well as the proportion of false negatives. Total accuracy is the total number of correct predictions divided by total observations. The proportion of false negatives is the total number of lakes that were predicted to not exceed the microcystin guidelines but actually did, divided by the total number of observations.\n\n\nResults\n\nIn the 2007 NLA, microcystin-LR concentrations ranged from 0.05 to 225 μg/L. Microcystin-LR concentrations of 0.05 μg/L represent the detection limits. Any value greater than that indicates the presence of microcystin-LR. Of those lakes with microcystin, the median concentration was 0.51 μg/L and the mean was 3.17 μg/L. Of all lakes sampled, 21% of lakes exceeded the US EPA child level, 8.8% of lakes exceeded the US EPA adult level, 11.7% of lakes exceeded the WHO drinking level, and 7.3% of lakes exceeded the WHO recreational level. For chlorophyll a, the range was 0.07 to 936 μg/L. All lakes had reported chlorophyll a concentrations that exceeded detection limits. The median concentration was 7.79 μg/L and the mean was 29.63 μg/L. The associations between chlorophyll a and the upper confidence interval across a range of conditional probability values are shown in Table 2. Specific chlorophyll a concentrations that are associated with greater than even odds of exceeding the advisory levels were 23.4, 67.0, 83.5, and 105.8 μg/L for 0.3, 1.0, 1.6, and 2.0 μg/L advisory levels, respectively (Table 2 & Figure 1).\n\nA. Plot for US EPA child (0.3 μg/L). B. Plot for WHO drinking (1 μg/L). C. Plot for US EPA adult (1.6 μg/L). D. Plot for WHO recreational (2 μg/L).\n\nThe chlorophyll a cutoffs may be used to predict whether or not a lake exceeds the microcystin-LR health advisories. Doing so allows us to compare the accuracy of the prediction as well as evaluate false negatives. Total accuracy of the four cutoffs predicting microcystin-LR exceedances were 75% for the US EPA children’s advisory, 86% for the WHO drinking water advisory, 89% for the US EPA adult advisory, and 91% for the WHO recreational advisory (Table 3–Table 6). However, total accuracy is only one part of the prediction performance with which we are concerned.\n\nWhen using the chlorophyll a cutoffs as an indicator of microcystin-LR exceedances, the error that should be avoided is predicting that no exceedance has occurred when in fact it has. In other words, we would like to avoid Type II errors and minimize the proportion of false negatives. For the four chlorophyll a cut-offs we had a proportion of false negatives of 9%, 7%, 6%, and 5% for the US EPA children’s, the WHO drinking water, the US EPA adult, and the WHO recreational advisories, respectively. In each case we missed less than 10% of the lakes that in fact exceeded the microcystin-LR advisory. While this method performs well with regard to the false negative percentage, it is possible that is a relic of the NLA dataset and testing with additional data would allow us to confirm this result.\n\n\nDiscussion\n\nThe association between Log10 microcystin-LR and Log10 chlorophyll a shows a wedge pattern (Figure 2). This indicates that, in general, higher concentrations of microcystin-LR almost always co-occur with higher concentrations of chlorophyll a yet the inverse is not true. Higher chlorophyll a is not necessarily predictive of higher microcystin-LR concentrations; however, chlorophyll a may be predictive of the probability of exceeding a certain threshold.\n\nThis is the case as the probability of exceeding each of the four tested health advisory levels increases as a function of chlorophyll a concentration (Figure 1). We used this association to identify chlorophyll a concentrations that are associated with a range of probabilities of exceeding a given health advisory level (Table 2). For the purposes of this discussion we focus on a conditional probability of 50% or greater (i.e., greater than even odds to exceed a health advisory level). The 50% conditional probability chlorophyll a thresholds represent 27.9%, 11.3%, 9%, and 6.9% of sample lakes for the US EPA child, the WHO drinking, the US EPA adult, and the WHO recreational levels, respectively.\n\nThere are numerous possible uses for the chlorophyll a and microcystin-LR advisory cut-off values. First, in the absence of microcystin-LR measurements, exceedance of the chlorophyll a concentrations could be a trigger for further actions. Given that there is uncertainty around these chlorophyll a cutoffs the best case scenario would be to monitor for chlorophyll a and in the event of exceeding a target concentration take water samples and have those samples tested for microcystin-LR.\n\nA second potential use is to identify past bloom events from historical data. As harmful algal blooms are made up of many species and have various mechanisms responsible for adverse impacts (e.g., toxins, hypoxia, odors), there is no single definition of a bloom. For cHABs, one approach has been to identify an increase over a baseline concentration of phycocyanin17. This is a useful approach for targeted studies, but phycocyanin is also not always available and measures the predominance of cyanobacterial pigments and not toxins. Using our chlorophyll a cutoffs provides a value that is more directly associated with microcystin-LR and can be used to classify lakes, from past surveys, as having bloomed.\n\nLastly, using chlorophyll a is not meant as a replacement for testing of microcystin-LR or other toxins. It should be used when other, direct measurements of cyanotoxins are not available. In those cases, which are likely to be common at least in the near future, using a more ubiquitous measurement, such as chlorophyll a will provide a reasonable proxy for the probability of exceeding a microcystin-LR health advisory level and provide better protection against adverse effects in both drinking and recreational use cases.\n\n\nData and software availability\n\nhttps://github.com/USEPA/microcystinchla\n\nhttp://dx.doi.org/10.5281/zenodo.4531718\n\nCreative Commons Zero 1.0: http://creativecommons.org/publicdomain/zero/1.0/",
"appendix": "Author contributions\n\n\n\nJH and BK conceived of and conducted the analysis. Both authors reviewed the results and contributed to writing the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to thank Anne Kuhn, Bryan Milstead, John Kiddon, Joe LiVolsi, Tim Gleason, and Wayne Munns for constructive reviews of this paper. This paper has not been subjected to Agency review. Therefore, it does not necessary reflect the views of the Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. This contribution is identified by the tracking number ORD-015143 of the Atlantic Ecology Division, Office of Research and Development, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency.\n\n\nReferences\n\nJetoo S, Grover VI, Krantzberg G: The Toledo drinking water advisory: Suggested application of the water safety planning approach. Sustainability. 2015; 7(8): 9787–9808. Publisher Full Text\n\nRinta-Kanto JM, Konopko EA, DeBruyn JM, et al.: Lake Erie Microcystis: Relationship between microcystin production, dynamics of genotypes and environmental parameters in a large lake. Harmful Algae. 2009; 8(5): 665–673. Publisher Full Text\n\nHarmful Algal Bloom and Hypoxia Research and Control Amendments Act: Harmful Algal Bloom and Hypoxia Research and Control Amendments Act of 2014. 2014; S. 1254. Reference Source\n\nMcElhiney J, Lawton LA: Detection of the cyanobacterial hepatotoxins microcystins. Toxicol Appl Pharmacol. 2005; 203(3): 219–230. PubMed Abstract | Publisher Full Text\n\nZurawell RW, Chen H, Burke JM, et al.: Hepatotoxic cyanobacteria: a review of the biological importance of microcystins in freshwater environments. J Toxicol Environ Health B Crit Rev. 2005; 8(1): 1–37. PubMed Abstract | Publisher Full Text\n\nUS EPA: Drinking water health advisory for the cyanobacterial microcystin toxins. EPA-820-R-15100. 2015. Reference Source\n\nWorld Health Organization: Cyanobacterial toxins: Microcystin-LR in drinking-water. Background document for development of WHO guidelines for drinking-water quality. Geneva, Switzerland. World Health Organization, 2nd ed. Geneva. 2003. Reference Source\n\nChorus I, Bartram J: Toxic cyanobacteria in water: A guide to their public health consequences, monitoring and management. World Health Organization. 1999. Reference Source\n\nJames R, Gregg A, Dindal A, et al.: Environmental technology verification report: Abraxis microcystin test kits. Online document. Accessed online: June 22, 2011. Reference Source\n\nAranda-Rodriguez R, Jin Z, Harvie J, et al.: Evaluation of three field test kits to detect microcystins from a public health perspective. Harmful Algae. 2015; 42: 34–42. Publisher Full Text\n\nPip E, Bowman L: Microcystin and algal chlorophyll in relation to nearshore nutrient concentrations in Lake Winnipeg, Canada. Environ Pollut. 2014; 3(2): p36. Publisher Full Text\n\nYuan LL, Pollard AI, Pather S, et al.: Managing microcystin: Identifying national-scale thresholds for total nitrogen and chlorophyll a. Freshwater Biol. 2014; 59(9): 1970–1981. Publisher Full Text\n\nUS EPA: National lakes assessment: A collaborative survey of the nation’s lakes. EPA 841-R-09-001. 2009. Reference Source\n\nPaul JF, Munns WR Jr: Probability surveys, conditional probability, and ecological risk assessment. Environ Toxicol Chem. 2011; 30(6): 1488–1495. PubMed Abstract | Publisher Full Text\n\nPaul JF, McDonald ME: Development of empirical, geographically specific water quality criteria: A conditional probability analysis approach. J Am Water Resour Assoc. 2005; 41(5): 1211–1223. Publisher Full Text\n\nHollister JW, Walker HA, Paul JF: CProb: a computational tool for conducting conditional probability analysis. J Environ Qual. 2008; 37(6): 2392–2396. PubMed Abstract | Publisher Full Text\n\nMiller TR, Beversdorf L, Chaston SD, et al.: Spatiotemporal molecular analysis of cyanobacteria blooms reveals Microcystis--Aphanizomenon interactions. PLoS One. 2013; 8(9): e74933. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHollister JW, Kreakie BJ: Associations between chlorophyll a and various microcystin-LR. Zenodo. 2016. Data Source"
}
|
[
{
"id": "12330",
"date": "22 Feb 2016",
"name": "Alan E. Wilson",
"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\nTitle and Abstract: For clarity, the authors might consider replacing “various” with “World Health Organization and U.S. Environmental Protection Agency”.\n\nArticle content: Using publicly available data produced from the 2007 USEPA National Lakes Assessment, the authors use conditional probability analysis to develop four chlorophyll concentration thresholds associated with two USEPA microcystin drinking water advisory targets, one WHO microcystin drinking water advisory target, and one WHO microcystin recreational use target. For most water resource managers, chlorophyll is much easier and cheaper to measure than microcystin. Given the threat that microcystins pose to human health, the purpose of this study is valuable. With that said, this study significantly overlaps with Yuan et al. (2014). The current study adds new advisory microcystin targets recently established by USEPA and uses a different statistical approach than Yuan et al. 2014. The authors cite relatively few studies. I think the authors need to more broadly consider the existing literature and describe how their findings relate to and build from past studies. Below, I provide some related studies that the authors might want to consider. I am certain that I have missed other relevant studies. Ahn et al. (2011)Beaver et al. (2014)Chan et al. (2007)Conti et al. (2005)Dolman et al. (2012)Downing et al. (2000)Giani et al. (2005)Graham et al. (2004)Graham et al. (2010)Jacoby et al. (2015)Kotak et al. (2000)Marion et al. (2012)Orihel et al. (2012)Sarnelle et al. (2010)Scott and Haggard (2015)Sinang et al. (2015)Stow et al. (2015)Su et al. (2015)Yuan and Pollard et al. (2015) Based on the 2007 National Lakes Assessment report, roughly two-thirds of the waterbodies reported no detectable microcystin (detection limit = 0.05 ug/L) despite covering a huge range of chlorophyll concentrations. And, Fig 2 suggests that a large number of sites had barely detectable concentration of microcystin across a wide range of chlorophyll. It is not clear from the text how the authors dealt with waterbodies with undetectable or barely detectable microcystin concentrations.\n\nPresenting histograms of chlorophyll and microcystin concentrations for the study lakes would be useful. I am not an expert on conditional probability analysis. Based on the authors’ text (second paragraph in Analytical Methods section), it appears that this analysis considers multiple events over time. If their dataset includes single measurements in a waterbody, I don’t understand where the temporal component comes into the analysis. Again, I could be totally misunderstanding how this analysis works and should probably read the relevant references the authors provided. Based on increasing error in the conditional probability plots as chlorophyll increases, the reported chlorophyll thresholds should not include significant digits (i.e., ± 0.1) but instead be whole numbers. I would organize the information in table 1 by either concentration (low to high) or advisory type (drinking or recreational) and concentration (low to high). It might also be useful to include the number of lakes represented in each category based on microcystin. In table 2, I would add the specific microcystin concentration target under each advisory type to avoid having to look back at table 1 for these data. Conclusions: The purpose of this study is to use a simple measurement (chlorophyll) to determine the threat that microcystins pose to a waterbody relative to existing microcystin concentration targets. Most waterbodies lacked microcystin and Figure 2 clearly shows that there are a huge number of waterbodies across a large chlorophyll range that apparently had microcystin concentrations at the detection limit of 0.05 ug/L. I am concerned about the microcystin data at the detection limit. They appear to be false positives. I agree with the authors who acknowledged that high chlorophyll is not always a good predictor of high microcystin. What should be done for those waterbodies with high concentrations of chlorophyll but that had no or barely detectable microcystin? Data: I am confused about the data collected and available for the 2007 National Lakes Assessment. For example, I organized this dataset in July 2010 and found that 1158 lakes were sampled once (1152 of these lakes included data for both chlorophyll and microcystin) and 95 of the 1158 originally sampled lakes were sampled a second time in 2007. Yuan et al. 2014 (Freshwater Biology) used data for 1077 sampled lakes. The current study (as well as the National Lakes Assessment website and report) describes data for 1028 lakes. Clarity about these discrepancies is not necessarily the authors’ job, but it would be good to understand why the differences exist across these datasets. Also, for this study, how were data used for lakes sampled twice in 2007?\n\nAlthough all of the National Lakes Assessment data are publicly available, the authors should provide the dataset that they used for this study.",
"responses": [
{
"c_id": "2005",
"date": "13 Jun 2016",
"name": "Jeffrey Hollister",
"role": "Author Response",
"response": "## I think the authors need to more broadly consider the existing literature and describe how their findings relate to and build from past studies. Below, I provide some related studies that the authors might want to consider. - **Response:** First thanks for the fantastic list of refs! Having it linked with this publication is a resource in and of itself. We have looked at those carefully and have added several: including Ahn et al, Beaver et al, Yuan and Pollard, and Marion et al. We have not added signficantly to the background on this paper because our goal was to keep this research communication short and focused on on the chl and microcystin relationship. ## Based on the 2007 National Lakes Assessment report, roughly two-thirds of the waterbodies reported no detectable microcystin (detection limit = 0.05 ug/L) despite covering a huge range of chlorophyll concentrations. And, Fig 2 suggests that a large number of sites had barely detectable concentration of microcystin across a wide range of chlorophyll. It is not clear from the text how the authors dealt with waterbodies with undetectable or barely detectable microcystin concentrations. - **Response:** We have added some text to the Data section indicating how we deal with the detection limit. We feel it is important to keep these values in the analysis as removing them would inflate our confidence around the conditional probabilities. We hope this is clearer in our revision. ## Presenting histograms of chlorophyll and microcystin concentrations for the study lakes would be useful. - **Response:** We have chosen to present the distribution information in text and present for both chlorophyll and microcystin the range, mean, and median. Figure 2 also indicates the distribution of both. Lastly, the data are availble via [code from the GitHub repository](https://github.com/USEPA/Microcystinchla/blob/master/R/get_nla.R). ## I am not an expert on conditional probability analysis. Based on the authors’ text (second paragraph in Analytical Methods section), it appears that this analysis considers multiple events over time. If their dataset includes single measurements in a waterbody, I don’t understand where the temporal component comes into the analysis. Again, I could be totally misunderstanding how this analysis works and should probably read the relevant references the authors provided. - **Response:** We have added some additional text in the methods about the NLA as well as in the Discussion on NLA limitations. In short, this is not a temporal analysis and is based on a single snap shot. ## Based on increasing error in the conditional probability plots as chlorophyll increases, the reported chlorophyll thresholds should not include significant digits (i.e., ± 0.1) but instead be whole numbers. - **Response:** Done. NEED TO DO on table already in overleaf ## I would organize the information in table 1 by either concentration (low to high) or advisory type (drinking or recreational) and concentration (low to high). It might also be useful to include the number of lakes represented in each category based on microcystin. - **Response:** Table re-orderd based on concentration. Number of lakes (as percentage) included in text. Need to do directly on table in overleaf. ## In table 2, I would add the specific microcystin concentration target under each advisory type to avoid having to look back at table 1 for these data. - **Response:** Done. Need to transfer to overleaf. ## Most waterbodies lacked microcystin and Figure 2 clearly shows that there are a huge number of waterbodies across a large chlorophyll range that apparently had microcystin concentrations at the detection limit of 0.05 ug/L. I am concerned about the microcystin data at the detection limit. They appear to be false positives. I agree with the authors who acknowledged that high chlorophyll is not always a good predictor of high microcystin. What should be done for those waterbodies with high concentrations of chlorophyll but that had no or barely detectable microcystin? - **Response:** We added some discussion about this in the last paragraph of the Data section. We feel that these should be left in as removing them would erroneously inflate our confidence intervals and impact the conditional probabilities. Essentially these are lakes with very low microcystin but widely varying chlorophyll values. ## I am confused about the data collected and available for the 2007 National Lakes Assessment. For example, I organized this dataset in July 2010 and found that 1158 lakes were sampled once (1152 of these lakes included data for both chlorophyll and microcystin) and 95 of the 1158 originally sampled lakes were sampled a second time in 2007. Yuan et al. 2014 (Freshwater Biology) used data for 1077 sampled lakes. The current study (as well as the National Lakes Assessment website and report) describes data for 1028 lakes. Clarity about these discrepancies is not necessarily the authors’ job, but it would be good to understand why the differences exist across these datasets. Also, for this study, how were data used for lakes sampled twice in 2007? - **Response:** We share your confusion! There are many \"types\" of samples included with the raw NLA data. For this analysis, we only used the probability samples (i.e. no reference samples) and only used the first visit to a lake. Additionally, lakes that had no data reported for either chl or microsystin were not included. As noted, this results in 1028 samples ## Although all of the National Lakes Assessment data are publicly available, the authors should provide the dataset that they used for this study. - **Response:** Code to access the data is available from [USEPA/microcysinchla](https://github.com/USEPA/microcystinchla). We have also added in a static .csv file to this repository of the data used for our analysis. This is listed in the \"Data and software availability\" section. Thank you for your review. We have just submitted our revisions and expect the new version to be available in the next few days. Below are our responses to the specific issues you raise. We feel the paper is stronger after this revision. Any questions, feel free to contact us or comment here. Thanks again! ## Specific Edits: ### Title and Abstract: - For clarity, the authors might consider replacing “various” with “World Health Organization and U.S. Environmental Protection Agency”. - **Response:** As this has already been indexed, we thought it best to limit the edits to the title."
}
]
},
{
"id": "12688",
"date": "05 Apr 2016",
"name": "Jason W. Marion",
"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\nOverview: The manuscript/article addresses a critical question applicable to recreational and drinking water managers: Can we rapidly predict potentially harmful cyanobacteria blooms using traditional water quality methods? This question is likely to become more relevant according to the most current literature as blooms are expected to increase in frequency in the midst of a warming global climate facing more extreme storm and drought events 1-6. Using the rather large and nationally applicable National Lakes Assessment database for the United States, the authors demonstrate some of the strengths and weaknesses of using chlorophyll a as an indicator for at-risk conditions that could warrant management action or follow-up testing for cyanotoxins. The authors are also able to assign action levels or at least share possible action levels for management action using conditional probabilities. The strengths and weaknesses of selected probabilities are described using analyses similar to specificity and sensitivity in the form of accuracy and ‘avoiding type II errors’. The authors do not propose using chlorophyll a as a proxy to replace toxin measurement, but as a tool to help facilitate targeted monitoring of toxins during at-risk conditions. Overall Comments: The article is meritorious in that it does provide a meaningful starting point for lakes with no phycocyanin measures and for providing a meaningful starting point for developing some semblance of an action level that could be employed by recreational water and drinking water managers concerned with cyanotoxins. The article does stand to improve significantly in some key areas, which are as follows: (1) Additional discussion in methods related to the National Lakes Assessment(2) Additional discussion needed on how the data were organized for data analysis(3) Improved discussion needed on alternative indicators for cHABs and cyanotoxins not assessed in the NLA(4) Consideration of region-specific criteria or limitations of national recommendations for chl a.(5) Greater discussion on limitations of NLA and need for model validation/future studies. (1) Additional discussion needed in methods related to the National Lakes Assessment:The readership may not be aware of the U.S. NLA performed in 2007. The author(s) should clarify where samples were collected (nearshore or from the surface in the deeper waters). NLA chlorophyll a samples were take from the profundal zone rather than the littoral zone. The readership may also be interested in how many chl a samples were collected from each lake. Where were the microcystin-LR samples collected? (2) Additional discussion needed on how the data were organized for data analysis:Were these samples paired (collected at the same time from the same locale) or are these some type of aggregated value over a lake season? Describing this in the methods will really help for understanding the importance of this work. Paired results (MC-LR and Chl a from the same day) are much more impactful for demonstrating the rapid advantage of chl a compated to using results that are a seasonal average indicating that the hypereutrophic and eutrophic lakes (ones with the highest chl a) are also the ones that are most likely to have a cyanoHAB event sometime during the year. (3) Improved discussion needed on alternative indicators for cHABs and cyanotoxins not assessed in the NLA: Brief mention is given to phycocyanin (one study), and the additional language (about phycocyanin not always being available for measure and when measured, it is for only measuring pigment and not toxins) is equally relevant for chl a. The same in vivo handheld fluorometers and continuous monitoring solutions available for chl a are now widely available for phycocyanin, often at the same cost as a rapid measure for chl a. Phycocyanin, like chl a, does not measure toxin either, but phycocyanin in many studies has outperformed chl a, and in some studies it has not (especially when toxin concentration is low). Historical records on PC are likely not as great as chlorophyll a. Overall, several studies on this topic have been produced in the last two to four years (see Zamyadi and Dorner’s work), with one study using phycocyanin to predict non-alcoholic liver disease presuming a relationship with cyanotoxins (Zhang et al. 2015) (4) Consideration desired on region-specific criteria or limitations of national recommendations for chl a: With nearly 30% of the lakes in the temperate plains being coded as poor for chlorophyll a in the 2007 NLA, what impact would these conditional probabilities have on these lakes? Should the lake managers in this region be monitoring continuously all the time? What are the mean/median chlorophyll a levels for this part of the U.S? Regional variability may be really important and did the conditional probability approach take this into consideration or can it take it into consideration? Is there a way to evaluate if there are significant regional effects in the U.S? For nutrient standards in the U.S. and macroinvertebrate assessments, EPA has had to issue region-specific guidelines/criteria, etc. for some parameters. (5) Greater discussion needed on limitations of NLA and need for model validation/future studies: The paper fails to address the limitations of the NLA – as a reader, I’m not aware of the limitations. I have much respect for the NLA, but I do have questions regarding the number of samples for each lake. Furthermore, a statement or two discussing the need to validate modeled data may be worthwhile. Is there a way to see if the probabilities actually align with the accuracy and type II error rates predicted by the conditional probability approach? Abstract-Specific Comments: Near the bottom of the abstract, the units seem quite high for microcystins (g/L) rather than micrograms/L. The micro Greek symbol (mu) may have been lost during uploading. Results Comments: (1) In discussing the lake exceedances of the various recommended levels by EPA, the addition of ‘drinking water’ is appropriate in my opinion. Although it is mentioned earlier in the methods, further providing the information in the results is helpful to a novice reader or a person just becoming familiar with drinking water regulations and guidelines, as the U.S. EPA child level may be presumed by a reader to be a level for recreation in a lake rather than a level associated with finished drinking water after water treatment. (2) “All lakes had reported chl a concentrations that exceeded detection limits” Does this mean that some were over range? Or does this mean that “All lakes had detectable levels of chl a” Discussion Comments: The wedge pattern in figure 2 is not apparent in figure 2, however, the logic makes sense and is supported visually by the conditional probability plots in fig 1. If figure 2 could have two lines of best fit (similar to the way some researchers do for funnel plots on publication bias papers), it may be easier to see the wedge shape. Climate Articles Highlighting Current Importance of Topic:Harvell, C. D.; Kim, K.; Burkholder, J. M.; Colwell, R. R.; Epstein, P. R.; Grimes, D. J.; Hoffman, E. E.; Lipp,E. K.; Osterhaus, A. D. M. E.; Overstreet, R. M.; Porter, J. W.; Smith, G. W.; Vasta, G. R.Emerging marine diseases – climate links and anthropogenic factors Science 2000, 285, 1505– 1510 Peperzak, L.Climate change and harmful algal blooms in the North Sea Acta Oecol. 2003, 24, 139–144 Edwards, M.; Johns, D. G.; Leterme, S. C.; Svendsen, E.; Richardson, A. J.Regional climate change and harmful algal blooms in the northeast Atlantic Limnol. Oceanogr. 2006, 51 ( 2) 820– 829 Ye, C.; Shen, Z.; Zhang, T.; Feng, M.; Lei, Y.; Zhang, J.Long-term joint effect of nutrients and temperature increase on algal growth in Lake Taihu, China J Environ. Sci. 2011, 23 ( 2) 222– 227 Paerl, H. W.; Hall, N. S.; Calandrino, E. S.Controlling harmful cyanobacterial blooms in a world experiencing anthropogenic and climatic-induced change Sci. Total Environ. 2011, 409, 1739– 1745 Davis, T. W.; Berry, D. L.; Boyer, G. L.; Gobler, C. J.The effects of temperature and nutrients on the growth and dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms Harmful Algae 2009, 8, 715– 725 Articles on Phycocyanin and Toxin Indicators Ought to Be Considered: Ahn, C.-Y.; Joung, S.-H.; Yoon, S.-K.; Oh, H.-M.Alternative alert system for cyanobacterial bloom, using phycocyanin as a level determinant J. Microbiol. 2007, 45 ( 2) 98– 104. Makarewicz, J. C.; Boyer, G. L.; Lewis, T. .; Guenther, W.; Atkinson, J.; Arnold, M.Spatial and temporal distribution of the cyanotoxin microcystin-LR in the Lake Ontario ecosystem: Coastal embayments, rivers, nearshore and offshore, and upland lakes J. Great Lakes Res. 2009, 35, 83– 89. Murby, A. L.Assessing spatial distributions of cyanobacteria and microcystins in N.H. lakes with implications for lake monitoring. Master’s Thesis. University of New Hampshire, 2009; p 89. Lehman, E. M.Seasonal occurrence and toxicity of Microcystis in impoundments of the Huron River, Michigan, USA Water Res. 2007, 41, 795– 802 Lee C, Marion JW, Cheung M, Lee CS, Lee J. Associations among Human-Associated Fecal Contamination, Microcystis aeruginosa, and Microcystin at Lake Erie Beaches. International journal of environmental research and public health. 2015 Sep 11;12(9):11466-85. Zamyadi A, Dorner S, Ndong M, Ellis D, Bolduc A, Bastien C, Prévost M. Application of in vivo measurements for the management of cyanobacteria breakthrough into drinking water treatment plants. Environmental Science: Processes & Impacts. 2014;16(2):313-23. Zhang F, Lee J, Liang S, Shum CK. Cyanobacteria blooms and non-alcoholic liver disease: evidence from a county level ecological study in the United States. Environmental Health. 2015 May 7;14(1):41.",
"responses": [
{
"c_id": "2004",
"date": "13 Jun 2016",
"name": "Jeffrey Hollister",
"role": "Author Response",
"response": "Thank you for your review. We have just submitted our revisions and expect the new version to be available in the next few days. Below are our responses to the specific issues you raise. We feel the paper is stronger after this revision. Any questions, feel free to contact us or comment here. Thanks again! ## Additional discussion needed in methods related to the National Lakes Assessment: The readership may not be aware of the U.S. NLA performed in 2007. The author(s) should clarify where samples were collected (nearshore or from the surface in the deeper waters). NLA chlorophyll a samples were take from the profundal zone rather than the littoral zone. The readership may also be interested in how many chl a samples were collected from each lake. Where were the microcystin-LR samples collected? - **Response:** We agree that additional information was needed describing the NLA. We have added this to the first paragraph on the Data section. ## Additional discussion needed on how the data were organized for data analysis: Were these samples paired (collected at the same time from the same locale) or are these some type of aggregated value over a lake season? Describing this in the methods will really help for understanding the importance of this work. Paired results (MC-LR and Chl a from the same day) are much more impactful for demonstrating the rapid advantage of chl a compated to using results that are a seasonal average indicating that the hypereutrophic and eutrophic lakes (ones with the highest chl a) are also the ones that are most likely to have a cyanoHAB event sometime during the year. - **Response:** We agree and have added some additional wording to the Data section indicating that the samples are taken at the same time. ## Improved discussion needed on alternative indicators for cHABs and cyanotoxins not assessed in the NLA: Brief mention is given to phycocyanin (one study), and the additional language (about phycocyanin not always beiThese blooms are expected to increase in frequecy and severity due to the impacts of climate change ng available for measure and when measured, it is for only measuring pigment and not toxins) is equally relevant for chl a. The same in vivo handheld fluorometers and continuous monitoring solutions available for chl a are now widely available for phycocyanin, often at the same cost as a rapid measure for chl a. Phycocyanin, like chl a, does not measure toxin either, but phycocyanin in many studies has outperformed chl a, and in some studies it has not (especially when toxin concentration is low). Historical records on PC are likely not as great as chlorophyll a. Overall, several studies on this topic have been produced in the last two to four years (see Zamyadi and Dorner’s work), with one study using phycocyanin to predict non-alcoholic liver disease presuming a relationship with cyanotoxins (Zhang et al. 2015) - **Response:** We agree that phycocyanin is more closely linked to microcystin than is chl *a*. Our paragraph mentioning phycocyanin was confusing and did suggest that chl *a* had a stronger association. Wording of that paragraph has been changed and the Ahn et al paper was added as reference. We feel that further discussion of phycocyanin, while important, is beyond the scope of our paper with its focus on chlorophyll. ## Consideration desired on region-specific criteria or limitations of national recommendations for chl a: With nearly 30% of the lakes in the temperate plains being coded as poor for chlorophyll a in the 2007 NLA, what impact would these conditional probabilities have on these lakes? Should the lake managers in this region be monitoring continuously all the time? What are the mean/median chlorophyll a levels for this part of the U.S? Regional variability may be really important and did the conditional probability approach take this into consideration or can it take it into consideration? Is there a way to evaluate if there are significant regional effects in the U.S? For nutrient standards in the U.S. and macroinvertebrate assessments, EPA has had to issue region-specific guidelines/criteria, etc. for some parameters. - **Response:** We agree that there are likely regional differences and would like to account for this; however, sample sizes for each region vary (67 to 155) and are relatively small. The resulting conditional probability analysis would have very wide confidence intervals. Thus, comparison between regions would be difficult and inferring a pattern would not be possible. We have added additional text in the discussion (second to last paragraph) that raises this issue. Additionally, we have added the Beaver et al. reference in this discussion. ## Greater discussion needed on limitations of NLA and need for model validation/future studies: The paper fails to address the limitations of the NLA – as a reader, I’m not aware of the limitations. I have much respect for the NLA, but I do have questions regarding the number of samples for each lake. Furthermore, a statement or two discussing the need to validate modeled data may be worthwhile. Is there a way to see if the probabilities actually align with the accuracy and type II error rates predicted by the conditional probability approach? - **Response:** We added a paragraph to the discussions about validation and the single sample limitations of the NLA. ## Specific Edits ### Abstract-Specific Comments: - Near the bottom of the abstract, the units seem quite high for microcystins (g/L) rather than micrograms/L. The micro Greek symbol (mu) may have been lost during uploading. - **Response:** Oops! Looks like it did occur during upload. Have double checked the final for the proper units. ### Results Comments: - In discussing the lake exceedances of the various recommended levels by EPA, the addition of ‘drinking water’ is appropriate in my opinion. Although it is mentioned earlier in the methods, further providing the information in the results is helpful to a novice reader or a person just becoming familiar with drinking water regulations and guidelines, as the U.S. EPA child level may be presumed by a reader to be a level for recreation in a lake rather than a level associated with finished drinking water after water treatment. - **Response:** Added this in. - “All lakes had reported chl a concentrations that exceeded detection limits” Does this mean that some were over range? Or does this mean that “All lakes had detectable levels of chl a” - **Response:** Changed wording to: All lakes had detectable levels of ... ### Discussion Comments: - The wedge pattern in figure 2 is not apparent in figure 2, however, the logic makes sense and is supported visually by the conditional probability plots in fig 1. If figure 2 could have two lines of best fit (similar to the way some researchers do for funnel plots on publication bias papers), it may be easier to see the wedge shape. - **Response:** Agreed that the wedge is not pronounced. We have changed the wording to better describe the pattern. Also, we disagree with the lines of best fit as those would then imply some sort of linear (presumably) pattern that we are not actually highlighting. Additionally the added lines would then require discussion and would detract from the focus on conditional probability."
}
]
},
{
"id": "12719",
"date": "11 Apr 2016",
"name": "Zofia E Taranu",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral commentsThis study provides an elegant framework to predict the severe impairment of U.S. lakes and reservoirs by cyanobacterial harmful algal blooms. I especially appreciated the clever use of conditional probability analysis to identify chlorophyll a threshold above which MC concentrations exceed WHO drinking water and recreational provisional guidelines. Chlorophyll a is a regularly measured water quality variable, and this study indeed offers a promising approach that can me used in many lakes around the world to identify problematic lakes or regions. In that regard, it would have been interesting to account for the spatial heterogeneity across this landscape -- as indicated by Beaver et al. (2014), some regions of the continental U.S. are more likely to be MC hotspots. Accounting for this heterogeneity will likely help explain some of the noise in the biplot shown in Fig. 2. I have also analyzed the MC data from the same dataset and found that accounting for different ecoregions in my model (as presented in Beaver et al., 2014) helped further explain the probability of detecting versus failing to detect MC in lakes. I am curious to know how this would play out with your modeling approach (conditional probability analysis). Minor commentsI recommend the following minor typographical corrections: P3Change: “Yuan et al. (2014) explore these associations in detail and control for other related variables. In their analysis they find that total [...]”To: “Recently, Yuan et al. (2014) explored these associations in detail and controlled for other related variables and found that total [...]” Change: “Given these facts, it should be possible to identify chlorophyll a concentrations that would be associated with the [...]”To: “These findings suggest that chlorophyll a concentrations could also track the [...]” Change: “Identifying these associations would provide another tool for [...]”To: “Identifying this association would provide an important tool for [...]” Change: “We add to past studies by exploring associations with newly announced advisory [...]”To: “We build on past studies by exploring associations with the newly announced advisory [...]” P4Change: “Thus, to identify chlorophyll a concentrations of concern we identify the value [...]”To: “Thus, to identify chlorophyll a concentrations of concern we identified the value [...]” Change: “were highly skewed right,”To: “were highly right skewed,” Change: “Lastly, we assess the ability of”To: “Lastly, we assessed the ability of” Change: “We use error matrices and calculate total accuracy”To: “We used error matrices and calculate total accuracy” Change: “For chlorophyll a, the range was”To: “Chlorophyll a ranged from”Please specify that this chlorophyll a range corresponds to a range from oligotrophic to hypereutrophic lakes. Change: “The associations between chlorophyll a and the upper confidence interval”To: “The association between chlorophyll a and the upper confidence interval” Figure 2 should first be presented in the Results section. Change: “This is the case as the probability of exceeding each of the four tested health advisory levels increases as a”To: “Indeed, the probability of exceeding each of the four tested health advisory levels increased as a” Change: “We used this association to identify chlorophyll a concentrations that are associated”To: “We used this association to identify chlorophyll a concentrations that were associated”",
"responses": [
{
"c_id": "2003",
"date": "13 Jun 2016",
"name": "Jeffrey Hollister",
"role": "Author Response",
"response": "Thank you for your review. We have just submitted our revisions and expect the new version to be available in the next few days. Below are our responses to the specific issues you raise. We feel the paper is stronger after this revision. Any questions, feel free to contact us or comment here. Thanks again! ## Spatial/Regional Heterogeneity in Chlorophyll/Microcystin realtionship We agree that there are likely regional differences and would like to account for this; however, sample sizes for each region vary (67 to 155) and are relatively small. The resulting conditional probability analysis would have very wide confidence intervals. Thus, comparison between regions would be difficult and inferring a pattern would not be possible. We have added additional text in the discussion (second to last paragraph) that raises this issue. Additionally, we have added the Beaver et al. reference in this discussion. ## Specific Edits ### P3 - Change: “Yuan et al. (2014) explore these associations in detail and control for other related variables. In their analysis they find that total [...]”, To: “Recently, Yuan et al. (2014) explored these associations in detail and controlled for other related variables and found that total [...]” - **Response:** Changed - Change: “Given these facts, it should be possible to identify chlorophyll a concentrations that would be associated with the [...]”, To: “These findings suggest that chlorophyll a concentrations could also track the [...]” - **Response:** Changed - Change: “Identifying these associations would provide another tool for [...]”, To: “Identifying this association would provide an important tool for [...]” - **Response:** Changed - Change: “We add to past studies by exploring associations with newly announced advisory [...]”, To: “We build on past studies by exploring associations with the newly announced advisory [...]” - **Response:** Changed ### P4 - Change: “Thus, to identify chlorophyll a concentrations of concern we identify the value [...]”, To: “Thus, to identify chlorophyll a concentrations of concern we identified the value [...]” - **Response:** Changed - Change: “were highly skewed right,”, To: “were highly right skewed,” - **Response:** Changed - Change: “Lastly, we assess the ability of”, To: “Lastly, we assessed the ability of” - **Response:** Changed - Change: “We use error matrices and calculate total accuracy”, To: “We used error matrices and calculate total accuracy” - **Response:** Changed - Change: “For chlorophyll a, the range was”, To: “Chlorophyll a ranged from” - **Response:** Changed - Please specify that this chlorophyll a range corresponds to a range from oligotrophic to hypereutrophic lakes. - **Response:** Changed - Change: “The associations between chlorophyll a and the upper confidence interval”, To: “The association between chlorophyll a and the upper confidence interval” - **Response:** Changed - Figure 2 should first be (Figure \\ref{fig:chla_micro_scatter})presented in the Results section. - **Response:** We did not changes this as we fill Figure 2 is not presenting results of our analysis but justifying the approach as is a better fit to be introduced in the Discussion. - Change: “This is the case as the probability of exceeding each of the four tested health advisory levels increases as a”, To: “Indeed, the probability of exceeding each of the four tested health advisory levels increased as a” - **Response:** Changed - Change: “We used this association to identify chlorophyll a concentrations that are associated”, To: “We used this association to identify chlorophyll a concentrations that were associated” - **Response:** Changed"
}
]
}
] | 1
|
https://f1000research.com/articles/5-151
|
https://f1000research.com/articles/5-1354/v1
|
13 Jun 16
|
{
"type": "Research Note",
"title": "Preliminary investigation of glyphosate resistance mechanism in giant ragweed using transcriptome analysis",
"authors": [
"Karthik Ramaswamy Padmanabhan",
"Kabelo Segobye",
"Stephen C. Weller",
"Burkhard Schulz",
"Michael Gribskov",
"Karthik Ramaswamy Padmanabhan",
"Kabelo Segobye",
"Stephen C. Weller",
"Burkhard Schulz"
],
"abstract": "Giant ragweed (Ambrosia trifida) is a highly competitive annual weed prevalent mainly in the United States across the eastern Corn Belt. Glyphosate has been a key herbicide to help tackle the spread of giant ragweed in the past few decades. Recently, there have been reports of widespread resistance to glyphosate in giant ragweed, with the mechanism of resistance yet to be determined. We designed a single-replicate RNA sequencing experiment to study the genes differentially expressed between glyphosate-resistant and glyphosate-sensitive biotypes of giant ragweed. We used a de novo assembly of the giant ragweed transcriptome to determine key marker genes that could help explain the mechanism of resistance.",
"keywords": [
"Herbicide resistance",
"giant ragweed",
"glyphosate",
"RNA-Seq"
],
"content": "Introduction\n\nGiant ragweed (Ambrosia trifida) is a problematic annual weed in the United States and Canada, particularly in fields where corn and soybean are grown1,2. Due to the extensive use of weed control measures such as the growth of genetically-modified glyphosate-resistant crops, giant ragweed populations have been kept in check3. But due to the overuse of glyphosate and strong selective pressure, resistant weeds have been reported across the world (http://www.weedscience.org)4,5. The mechanism of resistance to glyphosate in other common weeds such as Malaysian goosegrass, Italian ryegrass, and rigid ryegrass have been identified6–8. However, the glyphosate resistance mechanism in giant ragweed is still unknown. In this study, we compare gene expression differences between the glyphosate resistant and sensitive giant ragweed plants using a time course experiment and identify genes that could be involved in glyphosate resistance.\n\n\nMethods\n\nGlyphosate-resistant and glyphosate-sensitive seeds of giant ragweed were collected from Noble County, Indiana (N41° 28.470 W85° 29.371) and Darke County, Ohio (N40° 15.9 W84° 42.7) respectively. After seed germination, plants at the five-node growth stage were selected for herbicide treatment. mRNA was extracted from leaf disks following a protocol adapted from Eggermont et al. 2 cm diameter leaf disks from the first fully developed leaf were punched out, frozen in liquid nitrogen, and total RNA was extracted in a 2 ml test tube. mRNA was sequenced using Illumina TruSeq for four time points – pre-treatment (0 hour), 3 hours, 8 hours and 12 hours after treatment with glyphosate at a rate of 0.7kg ae ha-1 (recommended field rate) sprayed using a compressed-air bench top track sprayer with a nozzle pressure of 249 kPa delivering a volume of 187 L of spray solution ha-19. RNA sequences were assembled using the Trinity package (version r2012-10-05) from paired-end reads10.\n\nRNA-seq reads were mapped to the transcriptome assembly, and the counts per million transcripts (CPM) value was determined using RSEM (version 1.2.8)11. Since we observed clear systemic changes in gene expression even at the first time point, we used a set of genes previously published in rice analyses as controls to normalize the expression values12. The consistent expression of these genes with respect to each other across the time course was verified (Table 1). Gene level counts that were less than 1 CPM in all time points were excluded from further analysis. Expression ratios were then calculated for each assembly, comparing the expression levels in the glyphosate resistant and sensitive strains at each time point. Numbers larger than 1 therefore reflect genes (transcript assemblies) with higher expression in the resistant variety. Sampling variation was controlled by the addition of a pseudo count of 0.5 CPM before calculating expression ratios. Assemblies with expression ratios greater than 4, or less than -4 were considered to be differentially expressed and were further examined. Annotations of the transcriptome were done using Trinotate (version r2013-02-25)13.\n\n12 genes from the list of 25 genes identified by Jain (2009) showed relatively stable expression across all time points, and thus were used for determining the scaling factor for the normalization12.\n\n\nResults\n\nThere is a clear difference in gene expression patterns between the resistant and sensitive plants even before plants were treated with herbicide (Table 2). The top differentially expressed transcripts in resistant and sensitive plants before treatment are shown in Table 3 and Table 4. The response to glyphosate is rapid, and a large number of genes are significantly differentially expressed within the first three hours after treatment compared to pre-treatment expression levels (Table 5).\n\nThe number of genes that are expressed > 4-fold higher in glyphosate-resistant giant ragweed (Resistant +) or > 4-fold higher in glyphosate-sensitive giant ragweed (Sensitive +) are shown.\n\nGenes expressed higher in resistant plants tend to play important roles in pathogen response regulation.\n\nGenes expressed at a higher level in sensitive plants seem to impact control of stress response.\n\nAfter treatment with glyphosate, the number of differentially expressed genes increases rapidly within the first three hours, and continues to increase at later time points.\n\n+ at least four-fold higher expression level; = similar expression level;\n\n- at least four-fold lower expression level; PT post-treatment\n\nThe genes with at least a four-fold change in expression level were identified in resistant and sensitive plants, and pathways associated with significantly over-represented genes identified using agriGO (cutoff P < 1e-7)14,15. Pathways with terms such as “response to other organisms” and “lipid biosynthetic process”, both of which are known to be related to pathogen response, were the most significantly over-represented16. Contrastingly, pathways that are typically over-represented in the sensitive biotype are annotated with terms like “response to stress”, “response to oxidative stimulus” and “lignin biosynthesis”, which are known stress response indicators17. This leads us to speculate that, not only do resistant giant ragweed plants react to glyphosate treatment in a manner resembling pathogen defense reactions, but they are already primed by alterations in stress response processes to hyper-react. This is consistent with the rapid necrosis reaction observed in resistant giant ragweed biotypes used in this study.\n\n\nConclusion\n\nThe complete transcriptome assembly of giant ragweed has been deposited in the NCBI BioProject database (http://www.ncbi.nlm.nih.gov/bioproject/) and is publicly available under accession PRJNA267208. The preliminary time-course experiment presented here identified groups of genes that may explain glyphosate resistance in giant ragweed. A more extensive transcriptome analysis study, with multiple replicates of sensitive and resistant giant ragweed biotypes, from a broader range of geographic sources, and with shorter time intervals will be useful to overcome the limitations of this preliminary study.\n\n\nData availability\n\nThe transcriptome assembly is available in the NCBI BioProject database under accession PRJNA267208.\n\nF1000Research: Dataset 1. Raw data of glyphosate resistance mechanism in giant ragweed using transcriptome analysis, 10.5256/f1000research.8932.d12553018",
"appendix": "Author contributions\n\n\n\nSCW, BS and KS grew the giant ragweed plants in the greenhouse and extracted samples for RNA-seq. KRP and MG did the transcriptome assembly, annotation and expression analysis. KRP wrote the draft of the manuscript. All authors contributed to revision and discussion of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe study was completed thanks to grant number 207666 provided to Dr. Stephen C. Weller from the Trask Trust at Purdue University.\n\n\nAcknowledgements\n\nThe authors thank the Purdue Genomics Core Facility for performing the RNA sequencing.\n\n\nReferences\n\nAbul-Fatih HA, Bazzaz FA: The Biology of Ambrosia Trifida L.. II. Germination, Emergence, Growth and Survival. New Phytol. 1979; 83(3): 817–827. Publisher Full Text\n\nBassett IJ, Crompton CW: THE BIOLOGY OF CANADIAN WEEDS.: 55.: Ambrosia trifida L. Can J Plant Sci. 1982; 62(4): 1003–1010. Publisher Full Text\n\nHarrison SK, Regnier EE, Schmoll JT, et al.: Competition and fecundity of giant ragweed in corn. Weed Sci. 2001; 49(2): 224–229. Publisher Full Text\n\nDuke SO, Powles SB: Glyphosate: a once-in-a-century herbicide. Pest Manag Sci. 2008; 64(4): 319–25. PubMed Abstract | Publisher Full Text\n\nReddy KN, Norsworthy JK: Glyphosate-resistant crop production systems: impact on weed species shifts. Glyphosate Resist Crop weeds Hist Dev Manag. 2010. Publisher Full Text\n\nGomes MP, Smedbol E, Chalifour A, et al.: Alteration of plant physiology by glyphosate and its by-product aminomethylphosphonic acid: an overview. J Exp Bot. 2014; 65(17): 4691–703. PubMed Abstract | Publisher Full Text\n\nJasieniuk M, Ahmad R, Sherwood AM, et al.: Glyphosate-Resistant Italian Ryegrass (Lolium multiflorum) in California: Distribution, Response to Glyphosate, and Molecular Evidence for an Altered Target Enzyme. Weed Sci. 2008; 56(4): 496–502. Publisher Full Text\n\nPreston C, Wakelin AM, Dolman FC, et al.: A Decade of Glyphosate-Resistant Lolium around the World: Mechanisms, Genes, Fitness, and Agronomic Management. Weed Sci. 2009; 57(4): 435–441. Publisher Full Text\n\nEggermont K, Goderis IJ, Broekaert WF: High-throughput RNA extraction from plant samples based on homogenisation by reciprocal shaking in the presence of a mixture of sand and glass beads. Plant Mol Biol Report. 1996; 14(3): 273–279. Publisher Full Text\n\nGrabherr MG, Haas BJ, Yassour M, et al.: Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011; 29(7): 644–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi B, Dewey CN: RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011; 12: 323. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJain M: Genome-wide identification of novel internal control genes for normalization of gene expression during various stages of development in rice. Plant Sci. 2009; 176(5): 702–706. Publisher Full Text\n\nHaas BJ, Papanicolaou A, Yassour M, et al.: De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc. 2013; 8(8): 1494–512. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarris MA, Clark J, Ireland A, et al.: The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res. 2004; 32(Database issue): D258–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDu Z, Zhou X, Ling Y, et al.: agriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res. 2010; 38(Web Server issue): W64–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRaffaele S, Leger A, Roby D: Very long chain fatty acid and lipid signaling in the response of plants to pathogens. Plant Signal Behav. 2009; 4(2): 94–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu Y, Li WC, Xu YQ, et al.: Differential expression of candidate genes for lignin biosynthesis under drought stress in maize leaves. J Appl Genet. 2009; 50(3): 213–23. PubMed Abstract | Publisher Full Text\n\nPadmanabhan KR, Segobye K, Weller S, et al.: Dataset 1 in: Preliminary investigation of glyphosate resistance mechanism in giant ragweed using transcriptome analysis. F1000Research. 2016. Data Source"
}
|
[
{
"id": "14937",
"date": "01 Aug 2016",
"name": "Roland Beffa",
"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\nPreliminary investigation of glyphosate resistance in giant ragweed using transcriptome analysis from’ Padmanabhan is a very preliminary work, and the differentially expressed genes found have to be further validated, genetically or/and functionally. No isogenic populations of sensitive and resistant glyphosate plants were used. Whereas the paper is well written and the data clearly presented, this work is a good start, but it is in a too early phase to be indexed. The article might be, from my point of view, re-focused, on the transcriptome assembly and annotation. This would be acceptable.",
"responses": []
},
{
"id": "15735",
"date": "23 Aug 2016",
"name": "Elena Alvarez-Buylla",
"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\nGlyphosate is a widespread herbicide since 1994, when transgenic crops were commercially released into the environment. Several cases of glyphosate resistance have been reported, after repeated use of this herbicide, since then. Previous cases have included species of Poaceae or Amaranthaceae.\n\nThe manuscript by Padmanabhan and colleagues presents an interesting comparison between the transcriptomic profiles of giant ragweed plants collected from populations that are either resistant or sensitive to the use of glyphosate. This paper is relevant, because the authors investigate an agricultural weed that is not a Poaceae or Amaranthaceae. This contribution is useful to addressing if glyphosate resistance follows common metabolic pathways in distantly related plants. Moreover, they do so following an experimental set up, that can potentially be replicated and expanded; this is an additional contribution.\n\nWe summarize our main concerns on the present status of the work under review, and provide comments or suggestions that may help improve this work.\n\nA more comprehensive transcriptomic comparison would greatly contribute to the objetives of the work under review.\n\nWe recommend: Transcriptomic analyses made between resistant and sensitive ragweed individuals sampled at different time points during the experiment –which the authors summarize in Tables 3 and 4 and compare on table 5- with an explicit discussion of their results in light of the transcriptomic profiles (and highly expressed genes) described for other glyphosate-resistant tweeds, in order to discuss if common metabolic pathways that are not lineage specific, are being selected upon through pervasive use of glyphosate in the corn belt in the USA, or not.\n\nThe paper would also benefit from a more thorough discussion concerning the type and function of genes that were found to be highly expressed in resistant and/or sensitive giant ragweed biotypes. We recommend using other databases, such as KEGG. It would be particularly important to focus on genes involved in secondary growth, lateral meristem activity, or any aspect related to perennial habit, in contrast to non-perennial habit of close relatives, if there are any. Also, comparison to functional information available in other perennial and non perennial herbs that have evolved resistance, would be very useful.\n\nThe fact that the genes that are highly expressed in sensitive giant ragweed are those involved with chloroplast function, which is the function that is primarily and directly affected by glyphosate, through the inhibition of aromatic aminoacid synthesis. This is not addressed by the authors aside from Table 4. Such discussion would also improve the significance of the paper.\n\nIn the last paragraph of the discussion, the authors propose a hypothesis concerning the highly expressed genes in resistant ragweed. The following statement made by the authors is confusing and requires clarification: “This is consistent with the rapid necrosis reaction observed in resistant giant ragweed biotypes used in this study”. Necrosis is a common response to biotic and abiotic stress but this phrase could be further clarified if the authors maybe by explaining to the reader what is the main physiological response to glyphosate observed in resistant biotypes: necrosis of all leaves? Of some? Necrosis and then new leaf generation? Are any of these observed in non resistant biotypes, ever?\n\nIn the conclusions the authors suggest how a more thorough analysis could be carried on in the future for giant ragweed resistance. This made it clear that some details were missing in the methods section of the present paper: how many plants did they sample (two for each biotype for biological replicates; same individual plant, but two samplings for technical replicates?). These details should be clarified, please.\n\nA useful review article that may be cited and help tackle questions pertaining the possible metabolic pathways affected by glyphosate:\n\nThe Shikimate Pathway and Aromatic Amino Acid Biosynthesis in Plants Annual Review of Plant Biology Vol. 63: 73-105 (Volume publication date June 2012) DOI: 10.1146/annurev-arplant-042811-105439",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1354
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https://f1000research.com/articles/5-1353/v1
|
13 Jun 16
|
{
"type": "Research Note",
"title": "Decrease in rate of multiple sclerosis-related hospitalizations in Portugal",
"authors": [
"Marta Pereira",
"Dimitra Lambrelli",
"Sreeram V. Ramagopalan",
"Dimitra Lambrelli",
"Sreeram V. Ramagopalan"
],
"abstract": "We sought to investigate the rate of multiple sclerosis (MS)-related hospitalizations in Portugal and assess whether there have been temporal changes as described in other countries. Using data from the Portuguese National Discharge Registry, we observed that between 2008 and 2013 the rate of MS-related hospitalizations decreased by 44%, from 15.9/100 person-years (95% confidence interval (CI: 14.9-16.9) in 2008 to 8.9/100 person-years (95% CI: 8.2-9.6) in 2013. The change in hospitalization rates is in accordance with what has been observed in other countries, and coincides with the release of new therapies for MS in Portugal.",
"keywords": [
"multiple sclerosis",
"epidemiology",
"hospitalizations"
],
"content": "Introduction\n\nMultiple sclerosis (MS) is a chronic, neurodegenerative autoimmune disorder of the central nervous system1. With a prevalence of 1/800 in North America and Northern Europe, MS is the most common acquired neurological disorder in young adults1, posing a substantialburden on healthcare systems. Recent studies have, however, suggested that MS-related hospitalization rates have been declining over the last decades2,3.\n\nPortugal is considered a region of medium MS prevalence4, but epidemiological data are limited. In the present study, we sought to investigate the difference in the rate of MS-related hospitalizations in Portugal between 2008 and 2013.\n\n\nMethods\n\nData on the number of hospitalizations in 2008 and 2013 with an MS diagnosis [International Classification of Diseases, 9th revision (ICD 9): 340] were obtained from the National Hospital Discharge Registry, centrally held in the Portuguese Central Administration of the Health System. This database includes data about all hospitalizations in all public hospitals. In Portugal, the National Health Service provides universal access to healthcare and patients with MS are almost exclusively treated in public hospitals4. These years were chosen as MS prevalence data in 2008 and 2013 in Portugal were available from the Atlas of MS (http://www.msif.org/wp-content/uploads/2014/09/Atlas-of-MS.pdf)5. The total number of hospitalizations (1,177,048 in 2008 and 1,108,911 in 2013), as well as the estimates of the Portuguese population, were obtained from Portuguese official statistics6.\n\nThe incidence of hospitalizations (MS-related or all-cause hospitalizations) was calculated by dividing the number of hospitalizations in each year by the number of patients at risk in the same year. The Wald method was used to calculate 95% confidence intervals (CI). Two different definitions of MS-related hospitalizations were used: 1) where MS was the primary reason for admission — the MS ICD-9 diagnostic code was reported in the field of the primary diagnosis, and 2) where MS was reported as either a primary or a secondary reason for admission — the MS ICD 9 diagnostic code was in any position on the diagnoses fields (20 fields available). The proportion of MS-related hospitalizations in each year was obtained by dividing the number of MS-related hospitalizations in each year by the total number of hospitalizations in the same year. Demographic and clinical characteristics of patients with MS hospitalized in each year were described using number and percentage of patients for categorical variables, and mean and standard deviation (variables with a normal distribution) or median and interquartile range (variables with a skewed distribution) for continuous variables. Differences between characteristics in 2008 and 2013 were estimated using t-tests (variables with a normal distribution) or Mann–Whitney test (variables with a skewed distribution) for continuous variables, and Chi square test for other categorical variables. P values < 0.05 were considered to be statistically significant. Statistical analysis was performed using STATA software version 13.\n\n\nResults\n\nDemographic and clinical characteristics of patients with MS hospitalized in 2008 and 2013 are summarized in Table 1. Between 2008 and 2013, the incidence rate of MS-related hospitalizations decreased from 15.9/100 person-years (95% CI: 14.9–16.9) to 8.9/100 person-years (95% CI: 8.2–9.6), defined using only information recorded in the main diagnosis field, and from 25.5/100 person-years (95% CI: 24.4–26.8) to 19.4/100 person-years (95% CI: 18.5–20.4) using the main or secondary diagnoses respectively (Figure 1). In the same years, the incidence rate of all hospitalizations in Portugal decreased from 11.7/100 person-years (95% CI: 11.7–11.7) to 11.2/100 person-years (95% CI: 11.1–11.2) (Figure 1). The proportion of MS-related hospitalizations among all hospitalizations in Portugal decreased slightly from 0.07% to 0.05% between 2008 and 2013.\n\n* Described according to the groups of diseases defined in the International Classification of Diseases 9th revision MS, multiple sclerosis; SD, standard deviation; IQR, interquartile range\n\nThe age of the patients with MS hospitalized and the length of stay in the hospital increased significantly (Table 1) between 2008 and 2013, where MS was either only a primary diagnosis or a diagnosis anywhere on the patient record. There was an increase in the proportions of females admitted to hospital but this was only significant when MS was recorded in any of the diagnosis fields (Table 1; p value = 0.062 for MS as main diagnosis and p value = 0.006 for MS as main or secondary diagnosis).\n\nNo differences were observed in the proportion of patients that died during hospitalization (Table 1). Where MS diagnosis was anywhere on the patient record, the proportion of MS patients with other diagnoses during hospitalization increased significantly between 2008 and 2013 for all groups of diseases considered, except for infectious and parasitic diseases, neoplasms, diseases of the nervous system and sense organs (excluding MS), and congenital anomalies (Table 1).\n\n\nDiscussion\n\nHere we show a decrease in MS-related hospitalizations in Portugal from 2008 to 2013. Where MS was the primary diagnosis the decrease was substantial — approximately 44%. The rates of hospitalization observed in Portugal appear to be similar to that documented in Canada2,3, suggesting perhaps that the thresholds for admission are similar between the two countries. The change in hospitalization rates cannot be explained entirely by a general change in admissions in Portugal, although this did decline by 4%. The change in hospitalization coincides with the release of new therapies for MS, the first of which, natalizumab (Tysabri®), became available in June 2007 in Portugal7. It is plausible that these newer therapies prevented some hospital admissions for patients with MS. The average age of MS patients admitted in 2013 was older as compared to 2008. As MS prevalence has increased over the period, the decrease in hospitalization rate may reflect a diluting by more newly diagnosed patients with lower disease severity. There may also be a change in the way healthcare is delivered, with a possible shift to more outpatient services as seen in other countries. This may explain why the average length of stay for MS-related admissions has increased while the rate of admissions has decreased, although this change in disease management has generally preceded the time period we have investigated in other countries.\n\nLimitations of this study include the lack of clinical data and information on potential confounders. There are also the uncertainties associated with the prevalence data we had available to us. Despite these limitations, our results are in accordance with previous results on hospital admissions in patients with MS. Our findings provide further epidemiological data on MS in Portugal, healthcare resource use in these patients, and impetus to investigate other efforts to reduce hospitalizations in this population.\n\n\nData availability\n\nThe Portuguese Central Administration of the Health System provided the database and gave permission to use the data to describe in the article. The data are not available online, but are available upon request after approval of the study objectives.\n\nF1000Research: Dataset 1. Data of multiple sclerosis related hospitalizations in Portugal, http://dx.doi.org/10.5256/f1000research.8787.d1255898",
"appendix": "Author contributions\n\n\n\nMP performed the analysis. SVR contributed to the analysis and interpretation of the data. MP wrote the first draft and all authors contributed to subsequent drafts and the final paper.\n\n\nCompeting interests\n\n\n\nMP, DL and SVR are employees of Evidera.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors gratefully acknowledge the Portuguese Central Administration of the Health System for providing the MS-related hospitalization data.\n\n\nReferences\n\nRamagopalan SV, Dobson R, Meier UC, et al.: Multiple sclerosis: risk factors, prodromes, and potential causal pathways. Lancet Neurol. 2010; 9(7): 727–739. PubMed Abstract | Publisher Full Text\n\nMarrie RA, Elliott L, Marriott J, et al.: Dramatically changing rates and reasons for hospitalization in multiple sclerosis. Neurology. 2014; 83(10): 929–937. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEvans C, Kingwell E, Zhu F, et al.: Hospital admissions and MS: temporal trends and patient characteristics. Am J Manag Care. 2012; 18(11): 735–742. PubMed Abstract\n\nFigueiredo J, Silva Â, Cerqueira JJ, et al.: MS Prevalence and Patients' Characteristics in the District of Braga, Portugal. Neurol Res Int. 2015; 2015: 895163. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMultiple Sclerosis International Federation (MSIF): Atlas of MS 2013. Mapping Multiple Sclerosis around the World. 2013; Accessed February 2016. Reference Source\n\nInstituto Nacional de Estatística: Accessed February 2016. Reference Source\n\nSousa L, de Sa J, Sa MJ, et al.: The efficacy and safety of natalizumab for the treatment of multiple sclerosis in Portugal: a retrospective study. Rev Neurol. 2014; 59(9): 399–406. PubMed Abstract\n\nPereira M, Lambrelli D, Ramagopalan S: Dataset 1 in: Decrease in rate of multiple sclerosis-related hospitalizations in Portugal. F1000Research. 2016. Data Source"
}
|
[
{
"id": "14457",
"date": "21 Jun 2016",
"name": "João Correia de Sa",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Pereira and co-workers addresses an relevant issue: to bring to public some data about the hospitalization rates of MS patients in Portugal.\nThere are no methodological errors but, in my opinion, some changes should be introduced:\nAbout Introduction when the authors say that the epidemiological data in Portugal are limited I can´t agree. Portugal is a small country of about 10 million people and 3 prevalence studies and 1 incidence study published bring data enough for a knowledge of the basic epidemiological picture of MS in the country. There is of course other missing data (for instance mortality data/ burden of the disease for the society) but these are often missing in other countries. I considered changing saying for instance that though prevalence and incidence in MS in Portugal are now known, other epidemiological data about the disease are still lacking.\n\nThe author stay that they have found a \"decrease in MS related hospitalizations..... from 2008 to 2013\". In fact they have only these two dates for comparison. They have no data among these two years so that some variability can not be excluded.\n\nIn Portugal there are a great diversity of neurological care. Central Hospitals in Lisbon, Oporto and Coimbra treat MS in ambulatory but in other Towns: Évora,Beja, Leiria, Covilhã, Santarem, for example patients are admitted for treating relapses with Methyl, and for Natalizumab infusion because Day-Hospital units are not available. This should be taken in consideration in discussion and could certainly explain why there are so many short-time admissions",
"responses": []
},
{
"id": "14810",
"date": "06 Jul 2016",
"name": "Bruce V Taylor",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 report that documents a significant fall in the number of hospitalisations for MS in Portugal following the release of higher potency disease modifying drugs. The observation is interesting and adds some support to the presumed greater efficacy of these therapies. Although causality cannot be determined from such an observational study.\nThe finding that the age of admissions increased is also interesting as it may indicate that those with longer duration of disease (directly correlated with age) may have acquired more complications of MS for example urosepsis and have required greater admission rates.\n\nTo directly answer the question significantly more detail and prospective acquisition of data is required. however this report does provide a strong background rationale to undertake these studies and to acquire this type of data which is invaluable in assessing the benefits or otherwise of expensive and potentially higher risk therapies in MS.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1353
|
https://f1000research.com/articles/5-1336/v1
|
10 Jun 16
|
{
"type": "Data Note",
"title": "Whole-genome sequencing of nine esophageal adenocarcinoma cell lines",
"authors": [
"Gianmarco Contino",
"Matthew D. Eldridge",
"Maria Secrier",
"Lawrence Bower",
"Rachael Fels Elliott",
"Jamie Weaver",
"Andy G. Lynch",
"Paul A.W. Edwards",
"Rebecca C. Fitzgerald",
"Matthew D. Eldridge",
"Maria Secrier",
"Lawrence Bower",
"Rachael Fels Elliott",
"Jamie Weaver",
"Andy G. Lynch",
"Paul A.W. Edwards"
],
"abstract": "Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines—ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4—all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC.",
"keywords": [
"Esophageal adenocarcinoma",
"whole genome sequencing",
"cell line",
"high-grade dysplasia",
"cancer genome",
"copy number alteration",
"single nucleotide variant"
],
"content": "Introduction\n\nEsophageal adenocarcinoma (EAC), including cancers of the gastro-esophageal junction, represent a substantial health concern in Western countries due to its increasing incidence and poor prognosis. To date, there are no widely accepted animal models for EAC and a limited number of cell lines are all that are available for in vitro functional studies. Recent genome-wide sequencing projects have shown that EAC is one of the most highly mutated solid cancers with a high degree of heterogeneity (Dulak et al., 2013; Weaver et al., 2014). In addition to point mutations there are also widespread copy number alterations with evidence of catastrophic events such as chromothripsis and bridge fusion breakages in about one-third of cases (Nones et al., 2014). An accurate annotation of the mutational landscape of available EAC cell lines is therefore crucial for optimal experimental design, interpretation of genotype-phenotype data and to analyse drug sensitivities. We selected eight EAC cell lines—ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4—the identities of which have been verified by short tandem repeat (STR) analysis, p53 mutation and xenograft histology against the original tumors (Boonstra et al., 2010), and one esophageal high grade dysplasia (CP-D) cell line. We performed high-coverage paired-end whole genome sequencing and aligned the sequence data to the human reference genome in order to detect single nucleotide variants, indels and copy number alterations.\n\n\nMaterials and methods\n\nCell lines were obtained through commercially available repositories except JH-EsoAd1, which was a kind gift from Hector Alvarez (Table 1).\n\nVerified origin identifies cell lines whose pathological origin from EAC has been verified in Boonstra et al., 2010.\n\nAll cell lines were from a certified source (Table 1) and verified in house for >90% match with publicly reported STR profiles. Cell lines were mycoplasma tested and grown in standard conditions reported in cell repositories indicated in Table 1. Matched germline DNA was not available.\n\nGenomic DNA was prepared from cultured cells with AllPrepDNA/RNA Mini Kit (Qiagen) according to manufacturer’s instructions. A single library was created for each sample, and 90-bp paired-end sequencing was performed at Beijing Genomic Institute (BGI, Guangdong, China) according to Illumina (Ca, USA) instructions to a typical depth of 30×, with 94% of the known genome being sequenced to at least 10× coverage and achieving a Phred quality of 30 for at least 80% of mapping bases. FastQC 0.11.2 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) was used to assess the quality of the sequence data. Additional alignment, duplication and insert size metrics quality metrics are reported in Supplementary material 7. Sequence reads were mapped to the human reference genome (Ensembl GRCh37, release 84) using BWA 0.5.9 (Li, 2009), sorted into genome coordinate order and duplicates marked using Picard 1.105 (FixMateInformation and MarkDuplicates tools respectively, http://broadinstitute.github.io/picard). Original BAM files are available in the European Bioinformatics Institute (EBI) repository (project: PRJEB14018; sample accessions: ERS1158075-ERS1158083).\n\nGATK v3.2.2 (Broad Institute, MA, USA) was used to call and filter single nucleotide and indel variants compared to the reference genome. In brief, the steps run were as follows: 1) local realignment of reads to correct misalignments around indels using GATK RealignerTargetCreator and IndelRealigner tools; 2) recalibration of base quality scores using GATK BaseRecalibrator tool; 3) SNV and indel calling using GATK HaplotypeCaller which determines haplotype by re-assembly within regions determined to be active, i.e. where there is evidence for a variation, and uses a Bayesian approach to assign genotypes. Hard filters were applied to the resulting call set using recommendations available from the GATK documentation (https://www.broadinstitute.org/gatk) to generate a high-confidence set of SNV and indel calls. These were analyzed with Ensembl Variant Effect Predictor (release 75, http://www.ensembl.org/info/docs/tools/vep/index.html) to annotate with genomic features and consequences of protein coding regions (Supplementary material 4). For the purposes of the analysis, all variants with global minor allele frequency (GMAF) >0.0014 described in the 1000 Genomes project were separated out as likely germline polymorphisms (The 1000 Genomes Project Consortium et al., 2012) according to the criteria adopted in the Cosmic Cell Lines Project (Wellcome Trust Sanger Institute, Cambridge). Further, we removed all SNPs that have a minor allele frequency in the DBSNP (Ensembl v.58) and variants with a frequency ≥0.00025 in the ESP6500 (NHLBI GO Exome Sequencing Project, released June 20th 2012). A full list of the filtered variants is available in Supplementary material 4 and Supplementary material 6.\n\nCopy number (CN) analysis was carried out using Control-FREEC (Boeva et al., 2012). Control-FREEC computes and segments CN profiles and is capable of characterizing over-diploid genomes, taking into consideration the CG-content and mapability profiles to normalize read count in the absence of a control sample. Ploidy in each cell line was assessed interactively with the Crambled app v.2.0 according to the methods described by Lynch (2015).\n\n\nDataset validation\n\nWe identified a median of 1.3×105 variants across all 9 cell lines (range 105,487–151,879; Figure 1a, Table 2, Supplementary material 3, Supplementary material 4). We found that 1,5% of the variants were in coding regions; additionally, 4% fell in surrounding gene regions (i.e. regulatory as defined in Zerbino et al. (2015), upstream and downstream regions), 41% in introns and 23% in intergenic regions. Among the variants in the coding sequence, the majority, 57.4%, were in the UTR regions, followed by exonic missense and synonymous variants (21% and 11% respectively (Figure 1, Table 2, Supplementary material 3, Supplementary material 4). The number of variations identified in the high-grade dysplasia CP-D line was not significantly lower to the median of other EAC cell lines, consistent with the finding that such pre-malignant lesions have already accumulated many SNVs (Weaver et al., 2014). OACP4C and ESO26 showed the smallest and largest number of variants, respectively. (Figure 1, Table 2).\n\nA) Bar chart showing the distribution of called variants across various regions of the genome as indicated; B) Details of the coding sequence variants identified by the Variant Effect Predictor (Ensembl) expressed as a mean percentage value of all cell lines (values were not statistically different among samples).\n\nAbsolute number, median, median absolute deviation and range interval are listed for each category of mutation according to Variant Effect Predictor classification (Ensembl).\n\nA limitation of this study is represented by the lack of an available normal counterpart. In order to overcome this problem, in addition to the GATK calling pipeline we have applied a series of filters according to the criteria reported in methods and derived the 1000 Genomes Project (The 1000 Genomes Project Consortium et al., 2012), DBSNP (Ensembl v.58) and ESP6500 (released June 20th 2012). This approach reduced the number of variants by an order of magnitude from the original GATK pipeline (from a median of 4.1×106 to 1.3×105). Yet, the abundance of called variants compared to a range of 4,8×103-6×104 reported in human EAC (Weaver et al., 2014), may indicate that a proportion of the variants called in our final annotation are of germline origin. Also, additional mutations may have accumulated in vitro. A comprehensive annotation of the coding sequence variants identified is reported in Supplementary material 3 and Supplementary material 4.\n\nIn order to investigate how closely cell lines reflect the spectrum of mutations observed in human specimens we analysed the mutational landscape of known cancer and putative EAC driver genes and compared to the previously reported mutation rate (Dulak et al., 2013; Weaver et al., 2014; Figure 2b & 2c). 69% of EACs have TP53 mutations (Weaver et al., 2014), while all cell lines carried at least one deleterious TP53 mutation. A SMAD4 mutation was present in 2 of 9 cell lines, ESO26 and JH-EsoAd, consistent with the 13% observed in EAC (Weaver et al., 2014). We were not able to identify mutations in ARID1A (affected by UTR variants in 1 of 9 cell lines) that is reportedly mutated in about 10% of cases of EAC specimens. Only some of the missense variants in the genes shown in Figure 2b resulted in known pathogenic mutations (i.e. TP53, PIK3CA, and TLR4). Other genes harboured benign or likely benign variants and/or variants with uncertain functional significance.\n\nA) Log ratio of copy number status of the selected genes computed with Control-Freec (green indicates CN gain and red CN loss). Genome wide CN for each line is available in Supplementary material 1 and Supplementary material 3. B) SNVs identified by our pipelines and annotated by Variant Effect Predictor analysis (Ensembl). When more than one variant was present in a single gene, the most deleterious was annotated according to the color-coded legend reported at the bottom of the figure. A complete annotation of identified SNV are available in the Supplementary material 2. C) Blue and red bars indicate the mutation rate of EAC genes reported in Dulak et al., 2013; and Weaver et al., 2014, respectively.\n\nWe expanded our analysis to other cancer genes of potential relevance to OAC. We identified a pathogenic KRAS mutation in SKGT4, and a missense mutation of uncertain significance in MET (OE33), EGFR (CP-D, ESO26, IH-EsoAd1). Among DNA repair genes all cell lines carry benign missense variants of ATM and missense variants of uncertain significance in BRCA2. MSH2 is affected by a missense variant in SKGT4, splice site variants in CP-D, JH-EsoAd1, and UTR variants in ESO51 and OACP4 C (Supplementary material 3, Supplementary material 4, Supplementary material 6). Copy number analysis (Supplementary material 1, Supplementary material 2) identified recurrent amplifications in ERBB2, MYC, MET and SEMA5A, and deletions in SMAD4, CDKN2A, CCDC102B and SMARCA4.\n\nThis sequencing data will enable the research community to undertake and interpret further analyses (reviewed in Supplementary material 5) and to inform the use of these cell lines as a model of EAC. Our data highlight the need to develop additional in vitro models that have a germline reference genome to identify clearly the somatic changes (Gazdar et al., 1998). A larger number of cell lines might also more closely recapitulate the range of mutations observed in human disease.\n\n\nData availability\n\nBAM files are available at the European Nucleotide Archive (ENA, EMBL-EBI, www.ebi.ac.uk/ena, Study PRJEB14018). Accession numbers: CP-D ERS1158083; SK-GT-4 ERS1158082; OE33 ERS1158081; OACP4 C ERS1158080; OACM5.1 ERS1158079; JH-EsoAd1 ERS1158078; FLO-1 ERS1158077; ES051 ERS1158076; ES026 ERS1158075.",
"appendix": "Author contributions\n\n\n\nGC collected and analysed the data, ME, AGL, MS and LB carried out bioinformatic analysis, RFE and JW contributed to STR analysis and DNA preparation, RCF, PAWE and GC conceived the study and wrote the manuscript. RCF and PAWE obtained funding for the study.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by an MRC Programme Grant to R.C.F. and a Cancer Research UK grant to PAWE. The pipeline for mutation calling is funded by Cancer Research UK as part of the International Cancer Genome Consortium. G.C. is a National Institute for Health Research Lecturer as part of a NIHR professorship grant to R.C.F. AGL is supported by a Cancer Research UK programme grant (C14303/A20406) to Simon Tavaré and the European Commission through the Horizon 2020 project SOUND (Grant Agreement no. 633974).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary material 1. A) Copy Number Alteration of EAC cell lines according to ploidy shown by FREEC plots (loss, normal, and gain are indicated in blue, green and red, respectively). Genes annotated in red are the genes of the Cancer Genes Cosmic Census that fall in the amplified regions defined as copy number ≥5 for diploid and ≥7 for triploid and tetraploid cell lines. Genes annotated in blue are genes of the Cancer Genes Cosmic Census that fall in deleted regions with CN ≤1. B) Tables reporting all the genes of the Cancer Genes Cosmic Census that falls in deleted or amplified regions according to FREEC. Cell lines are shown in the following order 1) CP-D, 2) ESO26, 3) ESO51, 4) FLO-1, 5) JH-EsoAd1, 6) OACM5.1 C, 7) OACP4 C, 8) OE33, 9) SK-GT-4. Click here to access the data.\n\nSupplementary material 2. FREEC output of CNV by chromosome of the analysed cell lines. CNV of each cell line is indicated by chromosome consistently to known ploidy and in silico verification with the Crambled App (Lynch et al., 2015). Click here to access the data.\n\nSupplementary material 3. Effect Predictor Analysis annotated VCF files of GAKT called variants for CP-D, ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4 are available for download at the EMBL-EBI European Variation Archive (EVA, http://www.ebi.ac.uk/eva/) under the study PRJEB14018).\n\nSupplementary material 4. Filtered variants: 1) CP-D, 2) ESO26, 3) ESO51, 4) FLO-1, 5) JH-EsoAd1, 6) OACM5.1 C, 7) OACP4 C, 8) OE33, 9) SK-GT-4. Click here to access the data.\n\nSupplementary material 5.. Publicly Available datasets for analysed cell lines. For each cell line, currently available datasets from COSMIC, the Broad-Novartis Cancer Cell Line Encyclopaedia, and GEO (Gene Expression Omnibus) are listed. Click here to access the data\n\nSupplementary material 6.. Gitools readable file containing mutation calls for all genes. When more than one variant was present in a single gene, the most deleterious was annotated according to the color-coded legend reported at the bottom of the figure. Gitools is freely available for download at www.gitools.org (Perez-Llamas & Lopez-Bigas, 2011). Click here to access the data.\n\nSupplementary material 7. Alignment, duplication and insert size metrics for each cell line. Click here to access the data.\n\n\nReferences\n\nAltorki N, Schwartz GK, Blundell M, et al.: Characterization of cell lines established from human gastric-esophageal adenocarcinomas. Biologic phenotype and invasion potential. Cancer. 1993; 72(3): 649–57. PubMed Abstract | Publisher Full Text\n\nAlvarez H, Koorstra JB, Hong SM, et al.: Establishment and characterization of a bona fide Barrett esophagus-associated adenocarcinoma cell line. Cancer Biol Ther. 2008; 7(11): 1753–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoeva V, Popova T, Bleakley K, et al.: Control-FREEC: a tool for assessing copy number and allelic content using next-generation sequencing data. Bioinformatics. 2012; 28(3): 423–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoonstra JJ, van Marion R, Beer DG, et al.: Verification and unmasking of widely used human esophageal adenocarcinoma cell lines. J Natl Cancer Inst. 2010; 102(4): 271–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Both NJ, Wijnhoven BP, Sleddens HF, et al.: Establishment of cell lines from adenocarcinomas of the esophagus and gastric cardia growing in vivo and in vitro. Virchows Arch. 2001; 438(5): 451–6. PubMed Abstract | Publisher Full Text\n\nDulak AM, Stojanov P, Peng S, et al.: Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity. Nat Genet. 2013; 45(5): 478–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGazdar AF, Kurvari V, Virmani A, et al.: Characterization of paired tumor and non-tumor cell lines established from patients with breast cancer. Int J Cancer. 1998; 78(6): 766–74. PubMed Abstract | Publisher Full Text\n\nHughes SJ, Nambu Y, Soldes OS, et al.: Fas/APO-1 (CD95) is not translocated to the cell membrane in esophageal adenocarcinoma. Cancer Res. 1997; 57(24): 5571–8. PubMed Abstract\n\nLynch A: Crambled: A Shiny application to enable intuitive resolution of conflicting cellularity estimates [version 1; referees: 2 approved]. F1000Res. 2015; 4: 1407. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNones K, Waddell N, Wayte N, et al.: Genomic catastrophes frequently arise in esophageal adenocarcinoma and drive tumorigenesis. Nat Commun. 2014; 5: 5224. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPalanca-Wessels MC, Barrett MT, Galipeau PC, et al.: Genetic analysis of long-term Barrett’s esophagus epithelial cultures exhibiting cytogenetic and ploidy abnormalities. Gastroenterology. 1998; 114(2): 295–304. PubMed Abstract | Publisher Full Text\n\nPerez-Llamas C, Lopez-Bigas N: Gitools: analysis and visualisation of genomic data using interactive heat-maps. PLoS One. 2011; 6(5): e19541. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRockett JC, Larkin K, Darnton SJ, et al.: Five newly established oesophageal carcinoma cell lines: phenotypic and immunological characterization. Br J Cancer. 1997; 75(2): 258–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe 1000 Genomes Project Consortium, Abecasis GR, Auton A, et al.: An integrated map of genetic variation from 1,092 human genomes. Nature. 2012; 491(7422): 56–65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeaver JM, Ross-Innes CS, Shannon N, et al.: Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis. Nat Genet. 2014; 46(8): 837–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZerbino DR, Wilder SP, Johnson N, et al.: The ensembl regulatory build. Genome Biol. 2015; 16(1): 56. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "14325",
"date": "05 Jul 2016",
"name": "Ian Beales",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 examined the DNA sequences of 8 oesophageal adenocarcinoma cells lines and one high-grade dysplasia cell line, The authors should be congratulated for tackling this important unmet need in oesophageal cancer research and publishing these important findings in such an accessible manner. As the authors state, oesophageal adenocarcinoma seems to be one of the cancers carrying the most mutations, and although several cell lines, including those utilized in this study are commonly used for laboratory studies, there has never been a systemic study of the genetic abnormalities in these cells lines. The data in this study does fill that important gap, allowing comparisons between them and the cancer in vivo.\nThe methods are appropriate for the study and well-described and the abstract accurately represents the contents of the study. The results are appropriately and clearly presented. The conclusions appear to be sound based on the data presented and most importantly the paper provides the data to enable other researchers to build on these data and hopefully further refine laboratory models for oesophageal adenocarcinoma.",
"responses": []
},
{
"id": "14843",
"date": "11 Jul 2016",
"name": "Claire Palles",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 performed whole genome sequencing of eight esophageal adenocarcinoma cell lines and one esophageal high grade dysplastia cell line to an average depth of 30x. The authors have made the BAM and VCF files available through the EBI repository and this will be an excellent resource for researchers working on this cancer. We feel the methods used are appropriate and most of the analyses described are informative. We do however have a few suggestions for the authors to address, these are listed below:\n\nDataset validation WGS section:\nClarify the % of variants that fall in each sequence context, coding, intronic, regulatory, intergenic. We assume this should sum to 100%.\n\nIn the next sentence there is a “(“instead of a ” ,” “in front of the 21% and 11% respectively”\n\nTable 1: ploidy state of CP-D, should this be hypotetrapoid?\n\nParagraph 2 of this section: Change 4,8x103 to 4.8x103\n\nMuTect was used as variant caller in the Dulak paper and SomaticSniper was used in the Weaver paper. The authors should explain that they can’t use a somatic variant caller as these require a \"normal\" sample and also that application of a different caller for this cell line project may also make comparisons with the Dulak and Weaver papers less powerful.\n\nAnalysis of putative EAC driver genes:\nThere isn’t an ARID1A UTR variant shown for any of the cell lines in Figure 2b yet the authors mention 1 of the 9 cell lines has such a variant in the text.\n\nOn a related note we think the authors should consider the relevance of including UTR and synonymous changes in figure2b. We don’t think that these are considered in the Dulak and Weaver papers and are, as far as we understand, unlikely to be functional.\n\nSecond sentence of the second paragraph needs clarifying. Presumably missense mutations were found in MET and EGFR? IH-EsoAd1 should be JH-EsoAd1 in the same sentence.\n\nAuthors should make more of the fact that they have sequenced whole genomes whereas the COSMIC cell line project has only sequenced cell line exomes. The authors could perhaps highlight the useful extra data that is available from this sequencing effort, such as identification of mutations in putative regulatory regions and germline variants. Both classes of variants will be of interest to researchers working on understanding the genetics of oesophageal adenocarcinoma and wishing to identify appropriate cell models to work with.",
"responses": []
},
{
"id": "14746",
"date": "21 Jul 2016",
"name": "Marnix Jansen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Contino present their WGS analysis of 9 (verified) oesophageal adenocarcinoma cell lines. This is an adequate platform to present these data and the fact that the authors make all raw BAM files easily accessible to the community means that this study is particularly valuable to colleagues looking to contrast cell lines with particular genomic aberrations or different neo-antigenic burdens. Such studies always come with the known caveats of in vitro selection and the authors rightfully acknowledge this. As expected, the study in large part confirms earlier large scale sequencing studies of primary material. The lack of a patient-specific reference control means that the impact of more subtle genomic abnormalities in for example regulatory regions remain difficult to study. Nonetheless this work represents a valuable addition to previously published datasets and the authors are to be commended for publishing this analysis. The paper is terse and I enjoyed reading this study.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1336
|
https://f1000research.com/articles/5-363/v1
|
16 Mar 16
|
{
"type": "Case Report",
"title": "Case Report: Prune perineum syndrome: A rare case with an unfavourable outcome",
"authors": [
"Roberto I. Lopes",
"Francisco T. Dénes",
"Gustavo B. Messi",
"Marcos G. Machado",
"Francisco T. Dénes",
"Gustavo B. Messi",
"Marcos G. Machado"
],
"abstract": "Prune perineum syndrome (PPS) is a rare anomaly, with only two previous case reports, both dying in the perinatal period. We report the first case of PPS that reached childhood.The patient presented with hypoplastic genitalia and bilateral cryptorchidism. There was no evidence of an anal orifice. A significant prune-like mass was observed, extending from the perineum to both gluteal regions and to a cephalic mid-line bony prominence, with a 1cm central orifice that discharged urine. MRI confirmed the previous findings and revealed a right crossed ectopic kidney, intestinal malrotation, a hypoplastic infrarenal inferior vena cava and a hypoplastic right iliac artery. Endoscopic evaluation through the orifice revealed a cavity lined by urothelial mucosa, with a small communication to the anterior urethra in its anterior wall.\n\nA staged reconstruction was planned, with a first-step urinary diversion through a continent abdominal reservoir associated to bilateral orchiopexy. The patient was discharged from the hospital three weeks later under intermittent catheterization. The next planned surgical step was the resection of the perineal mass and its cavity associated to the removal of the prominent sacrococcygeal bones. Unfortunately, four months after the first surgery the patient developed an acute abdomen and underwent a laparotomy that revealed a necrotic ileal segment secondary to obstructive adherences. He developed severe malabsorption followed by septic shock, dying five weeks after the procedure.\n\nDue to the lack of literature, there is no consensus for the management of these cases. The wish of the family for a better quality of life and social acceptance, compelled us to perform a urinary diversion, to be followed by a plastic and orthopedic reconstruction. Despite the successful initial result, the patient developed a late abdominal obstruction that was misdiagnosed, precipitating his untimely death five months after the first procedure.",
"keywords": [
"Prune perineum syndrome",
"pelvic malformations",
"congenital disease",
"urinary tract"
],
"content": "Introduction\n\nPrune perineum was first described in 1979 by Peeden et al.1 and to our knowledge only one further case has been presented2. PPS includes significant abnormalities in the development of the caudal axis, with characteristic deformation of the sacrococcygeal spine associated with a large wrinkled perineal mass and a hypoplastic external genitalia. In both previously described cases, the patient died in the perinatal period1,2. We present the first case of a male born with this disease who reached 5 years of age.\n\n\nCase report\n\nThe patient was born at the 39th week of the uneventful first gestation of a 28-year-old woman, without any family history of congenital malformation or chronic illness. The maternal medical history was uneventful and pregnancy was normal until the last trimester, when control ultrasonography disclosed a fetal perineal abnormality, therefore a cesarean delivery was performed. A large perineal mass, associated to imperforated anus was readily identified. Urine was observed leaking through an orifice in the perineal mass. A left flank terminal colostomy was performed on the second day of life. Post-operatively, the patient presented with good clinical course, with functioning colostomy and preserved renal function, being discharged after 2 weeks. At four years of age, he came to our service without any previous significant clinical problems, including urinary tract infection. The somatic growth and neuro-psycho-motor development were normal. He had no medication, was wearing diapers and a colostomy bag. Despite a small limp, he was able to walk normally.\n\nAt physical examination, all abnormalities were below the umbilicus. The abdomen had a functioning left colostomy while the genital area presented hypoplastic male external genitalia with bilateral impalpable cryptorchidism. There was no evidence of an anal orifice. The right leg was shorter than the left (Figure 1). Posteriorly, a 20 × 15 × 8 cm prune-like mass was observed, extending from the perineum to both gluteal regions and to a cephalic mid-line bony prominence, with a 1cm central orifice that discharged urine. The corrugated skin of the prune-like mass could be easily lifted, disclosing, through its orifice, a large cavity below that contained urine (Figure 2). In the sitting position, the mass and its cavity were compressed against the perineum.\n\nHypoplastic external genitals in detail.\n\nPosterior view showing perineal mass with central orifice in detail (A). Lateral view (B), and perspective (C).\n\nLaboratory examination showed a normal blood count and renal function, and the karyotype was 46, XY. Pelvic radiographs demonstrated an exaggerated sacrococcygeal concavity, lumbar dysrrhaphism and pubic dyastasis. The dorsal bony prominence corresponded to the sacrococcygeal spine, whose abnormal concavity oriented its tip upward, like a small tail. The contrast injection through the orifice of the mass showed a large cavity that extended sagittally and laterally. MRI confirmed the previous findings and revealed a right crossed ectopic kidney, intestinal malrotation, a hypoplastic infrarenal inferior vena cava and a hypoplastic right iliac artery. The posterior concavity of sacrococcygeal bones, associated to a deformity of the pelvic rim was evident, as well as the cavity below the convoluted skin and subcutaneous layer of the perineal mass (Figure 3). The endoscopic evaluation through the orifice revealed a cavity lined by urothelial mucosa, with a small communication to the anterior urethra in its anterior wall. The ureteral orifices were not identified, although urine accumulated in the cavity during the examination.\n\nNuclear magnetic resonance demonstrating exaggerated sacrococcygeal convexity (A); crossing ectopic right kidney and an intestinal malrotation (B); absence of pubic bone, and the corrugated skin covering the posterior perineal cavity (C).\n\nAfter extensive evaluation of the case and considering the psychological effects of the malformation, as well as the parental wish for a better quality of life and social acceptance of the child, we planned a staged reconstruction, with a first-step urinary diversion through a continent abdominal reservoir associated to bilateral orchiopexy. The procedure was successful and the patient recovered uneventfully. He was discharged from the hospital three weeks later with a dry perineal cavity and under intermittent catheterization regularly performed by the mother. The next surgical step would be the resection of the perineal mass and its cavity associated to the removal of the prominent sacrococcygeal bones. Unfortunately, four months after the first surgery the patient developed an acute abdomen that was misdiagnosed and treated as a urinary tract infection elsewhere. Forty-eight hours later, he was transferred to our service and was submitted to exploratory laparotomy that revealed a necrotic ileal segment secondary to obstructive adherences. The resection of the necrotic segment associated to a terminal ileostomy was performed, but he developed severe malabsorption followed by septic shock, dying five weeks after the procedure.\n\n\nDiscussion\n\nPreviously, only two cases of the rare prune perineum syndrome have been described, and both patients died in the perinatal period due to sepsis1,2. Early colostomy and normal renal function by an unobstructed solitary kidney enabled long-term survival in our case. Contrary to the two previous cases, this was a male patient with a 46 XY karyotype, although with a very small penis and impalpable testes.\n\nDue to the lack of literature, there is no consensus for the management of these unfortunate children. We report the unsuccessful outcome of a child treated for this syndrome. Even though he presented a quite stable physical development without any neurological impairment up to the age of four years, the wish of the family for a better quality of life and social acceptance, compelled us to perform a urinary diversion, to be followed by a plastic and orthopedic reconstruction. Despite the successful initial result, the patient developed a late abdominal obstruction that was misdiagnosed, precipitating his untimely death five months after the first procedure.\n\n\nConsent\n\nWritten informed consent was obtained from the parent of the patient for publication of this case report and any accompanying images and/or other details that could potentially reveal the patient’s identity.",
"appendix": "Author contributions\n\n\n\nRIL and GBM retrieved patient data, reviewed the literature and wrote the manuscript. MGM and FTD did a critical revision of the manuscript for scientific and factual content. All authors approved the final version 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\nPeeden JN Jr, Wilroy RS Jr, Soper RG: Prune perineum. Teratology. 1979; 20(2): 233–236. PubMed Abstract | Publisher Full Text\n\nWilliams DA, Weiss T, Wade E, et al.: Prune perineum syndrome: report of a second case. Teratology. 1983; 28(1): 145–148. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13606",
"date": "06 May 2016",
"name": "Dana Weiss",
"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 very interesting and unique case report.Some questions that arise:Had the child had any renal imaging after birth? Was the urine leaking through a penile urethra as well as through the orifice that contained urine? This part is not clear from the description. The authors could assist the reader additionally, should this case ever arise again, by describing exactly what kind of reconstruction was done at the first stage. Was a true bladder found? Were the ureters implanted normally into a bladder? Was there a bladder neck and urethra? What exactly was the “orifice” that was draining urine? When the authors say they created a continent abdominal reservoir – was this a bowel reservoir to which the ureters were implanted? If there was any bladder present, was this used? This kind of case report is interesting and important since many pediatric surgeons and urologists may never see something like this in our lifetime, but if we do, it is very valuable to have some inkling of what is going on, and how we might even begin to manage it.",
"responses": [
{
"c_id": "1974",
"date": "10 Jun 2016",
"name": "Roberto Iglesias Lopes",
"role": "Author Response",
"response": "Author Response to Reviewer's Comments:Had the child had any renal imaging after birth?Answer: He was referred to us at age 4, having undergone previously a colostomy elsewhere on the second day of life. We started all diagnostic workup from point zero. Was the urine leaking through a penile urethra as well as through the orifice that contained urine? Answer: The perineal orifice communicated to a cavity, where we identified a bladder, which was abnormally located perineally. The orifice was at least 1 cm large, and all urine leaked through this orifice. Nevertheless, on cystoscopic evaluation, a guidewire was inserted through the urethra and reached the bladder (the urethra was patent, but very thin).Was a true bladder found?Answer: Besides the large perineal cavity (identified as bladder) drained by the orifice and connected to the urethra, there was no other structure resembling a normal bladder. Were the ureters implanted normally into a bladder? Answer: As urine was continuously flowing into the perineal cavity (bladder) and leaking through the perineal cavity, it was evident that the ureter (draining alone the right crossed ectopic kidney) reached the perineal cavity. Nevertheless, on \"cystoscopic\" evaluation we weren't able to find the ureteral orifice. Was there a bladder neck and urethra? Answer: As mentioned before, a patent, albeit very thin urethra was connected between the perineal cavity (bladder) and the hypoplastic penile shaft. The bladder neck was also very small. What exactly was the “orifice” that was draining urine? Answer: We do not have an embryologic explanation for it, and we can only postulate that a fistula eventually ruptured between the \"perineal bladder\" and skin before birth due to increased intravesical pressure (the perineal wrinkled skin was very thin and had no bony support). When the authors say they created a continent abdominal reservoir – was this a bowel reservoir to which the ureters were implanted? Answer: Exactly: it was a continent bowel reservoir, but only one ureter was implanted to it, as the patient had only one kidney. If there was any bladder present, was this used? Answer: The presumptive primitive bladder, namely the perineal cavity, was left in situ, for posterior removal. It became completely dry after surgery."
}
]
},
{
"id": "12915",
"date": "20 May 2016",
"name": "Rodrigo L.P. Romao",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a case of prune-perineum, an extremely rare malformation for which survival beyond the perinatal period has not been documented. Only 2 previous case reports are described in the literature.\n\nThe description of the findings is interesting and the illustrations are of good quality. The description of the surgical technique is short. If the authors were able to provide more details about the reconstruction performed, it would be of interest to the reader. It is not clear what kind of reservoir was created and how exactly CIC was conducted postoperatively. A detailed account of the findings and intra-operative decision-making process would strengthen the report significantly.\nThe authors should be applauded for reporting the best outcome thus far for this exceedingly rare and complex condition.",
"responses": [
{
"c_id": "2002",
"date": "10 Jun 2016",
"name": "Roberto Iglesias Lopes",
"role": "Author Response",
"response": "If the authors were able to provide more details about the reconstruction performed, it would be of interest to the reader. It is not clear what kind of reservoir was created and how exactly CIC was conducted postoperatively. A detailed account of the findings and intra-operative decision-making process would strengthen the report significantly. Answer: A perineal orifice communicated to a cavity, where we identified a bladder, which was abnormally located perineally. The orifice was at least 1 cm large, and all urine leaked through this orifice. Nevertheless, on cystoscopic evaluation, a guidewire was inserted through the urethra and reached the bladder (the urethra was patent, but very thin). A patent, albeit very thin urethra was connected between the perineal cavity (bladder) and the hypoplastic penile shaft. The bladder neck was also very small. As the bladder consisted of a cavity lined with urothelium and was not a true bladder, we opted for a continent bowel reservoir. Only one ureter was implanted to it, as the patient had only one kidney. A true bladder was not available and therefore, a continent cutaneous ileal neobladder was performed, with the appendix used for clean intermittent catheterization every four hours in the postoperative care."
}
]
},
{
"id": "14064",
"date": "31 May 2016",
"name": "Andrew J. Kirsch",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 fascinating case of Prune Perineum Syndrome - the first case of survival beyond infancy. Only two others have been reported with PPS over the past half century. The experienced group of pediatric surgeons describe this exceedingly rare condition and offer the child an opportunity to improve quality of life through surgical reconstruction. The preoperative imaging studies are highly educational and instructive. Unfortunately, a delayed postoperative complication and poor access to emergency medical care lead to the patient’s demise. This report, however, provides important clinical information to the limited available literature on this condition. This case report will help to educate and guide future generations of patients and their families, as well as physicians and surgeons in the diagnosis and management of PPS.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-363
|
https://f1000research.com/articles/5-1326/v1
|
10 Jun 16
|
{
"type": "Review",
"title": "Mitral valve repair",
"authors": [
"Alberto Pozzoli",
"Michele De Bonis",
"Ottavio Alfieri",
"Michele De Bonis",
"Ottavio Alfieri"
],
"abstract": "Mitral regurgitation (MR) is the most common valvular heart disease in the Western world. The MR can be either organic (mainly degenerative in Western countries) or functional (secondary to left ventricular remodeling in the context of ischemic or idiopathic dilated cardiomyopathy). Degenerative and functional MR are completely different disease entities that pose specific decision-making problems and require different management. The natural history of severe degenerative MR is clearly unfavorable. However, timely and effective correction of degenerative MR is associated with a normalization of life expectancy. By contrast, the prognostic impact of the correction of functional MR is still debated and controversial. In this review, we discuss the optimal treatment of both degenerative and functional MR, taking into account current surgical and percutaneous options. In addition, since a clear understanding of the etiology and mechanisms of valvular dysfunction is important to guide the timing and choice of treatment, the role of the heart team and of echo imaging in the management of MR is addressed as well.",
"keywords": [
"Mitral valve",
"Mitral regurgitation",
"Transesophageal echocardiography",
"Percutaneous intervention"
],
"content": "Introduction\n\nMitral regurgitation (MR) is the most frequent clinically recognizable valvular heart disease in the Western world1. MR can be divided into organic MR, resulting from primary anatomical changes of the leaflets and subvalvular apparatus, or functional (secondary) MR, which is a consequence of annular dilatation and geometrical distortion of the subvalvular apparatus secondary to left ventricular (LV) remodeling and dyssynchrony, most usually associated with cardiomyopathy or coronary artery disease (mitral valve [MV] is morphologically normal). Organic and functional MR are different entities with regard to pathophysiology, prognosis, diagnosis, and management and will be discussed separately in this review. The most common etiology of organic MR in industrialized countries is degenerative MV disease, as a result either of myxomatous degeneration or of fibroelastic deficiency of the leaflets, leading to MV prolapse. Less common is organic MR due to rheumatic heart disease (prevalent in developing countries) and congenital MV anomalies2. Functional MR worsens the prognosis of patients with dilated cardiomyopathy3,4. Ischemic MR is a subcategory of functional MR in which LV dysfunction is the consequence of a previous myocardial infarction. The natural history of severe MR is clearly unfavorable, leading to LV failure, pulmonary hypertension, atrial fibrillation (AF), stroke, and death5. Appropriate and timely correction of degenerative MR, however, has a highly beneficial impact on the prognosis of patients and can even be associated with a life expectancy and a quality of life similar to those of the general population. For functional MR, surgery is challenging and outcomes are inferior to those of degenerative MR, and the indications and choice of technique are not supported by robust evidence6.\n\nFurthermore, in recent years, a variety of approaches for the percutaneous treatment of MR have emerged. The most widely adopted has been the edge-to-edge (EE) procedure investigated in large registries and small randomized trials. Meanwhile, numerous alternative technologies are in development.\n\n\nThe heart team\n\nA multidisciplinary heart team (interventional cardiologists, cardiac surgeons, anesthesiologists, imaging, and heart failure specialists) should evaluate the advantages and the drawbacks of surgical, percutaneous, and conservative approaches in all high-risk subjects with MR, assessing the risk ratio due to the presence of relevant comorbidities. The team should assess the possible futility of intervention in very high-risk patients, in whom conservative management could be more appropriate. Risk assessment is fundamental to decision-making since percutaneous MV intervention should currently be reserved for high-risk or inoperable patients. Definitions of ‘high surgical risk’ and the ‘inoperable patient’ remain elusive and significantly influenced by surgeon and center experience. Established risk scores—for example, Society of Thoracic Surgeons (STS) and EuroScore II—should be used in conjunction with other factors (e.g. frailty, porcelain aorta, and so on) as recommended by the MVARC (Mitral Valve Academic Research Consortium) consensus documents7,8. As heart valve teams generate the most beneficial treatments, it becomes fundamental to define the “centers of excellence” in MV repair. Criteria should include MV surgery volume requirements (center and surgeon), appropriate periprocedural imaging skills, and a willingness to provide patients and referring doctors with the data regarding outcomes based on the experience of the institution (including repair rates, mortality rates, stroke rates, and the likelihood of durability of the repair)9. A tailored approach for individual patients remains appropriate in the absence of guidelines for the conduct of heart team activity.\n\n\nImaging assessment\n\nTransesophageal echocardiography (TEE) is essential to understand MV anatomical morphology (leaflet, annular, and subvalvular anomalies), and it is fundamental to assess the degree of MR (Table 1 and Figure 1), defining suitability for an optimal surgical or percutaneous MV repair10. Moreover, the presence of thrombi in heart chambers or active endocarditic lesions, which could contraindicate intervention, should be detected. In particular for surgical patients with degenerative MR, both the leaflets and the corresponding associated lesions should be recognized and carefully studied. The management of asymptomatic patients is controversial as there are no randomized trials to support any particular course of action. Surgery can be proposed in selected asymptomatic patients with severe MR, in particular when repair probability is high. Some specific triggers for early intervention in these patients are worth mentioning: signs of LV dysfunction—in particular, LV ejection fraction (EF) of not more than 60% or LV end-systolic diameter (LVESD) of at least 45 mm or both—and lower LVESD are accepted in patients of small stature. If LV function is preserved, new-onset AF or pulmonary hypertension (at least 50 mmHg at rest or at least 60 mmHg during exercise) or both and sinus rhythm with severe LA dilatation (volume index of at least 60 ml/mq body surface area) provide surgical indication6. The detection of annular calcification is a finding of paramount importance. Instead, for surgical patients presenting with secondary MR, echocardiographic parameters on the LV morphology and function (volume, EF, and sphericity index) are dominant in association with geometric MV distortion (tenting area, coaptation depth, leaflet angles, and inter-papillary muscle distance). Numerous predictors of MV repair failure after undersized annuloplasty have been identified (Figure 2) and, when present, should suggest MV replacement as a more durable solution10.\n\n*Nyquist limit 50–60 cm/s. **Average between apical four- and two-chamber views. ***In the absence of mitral stenosis or other causes of elevated left atrial pressure. EROA, effective regurgitant orifice area.\n\nLV, left ventricular; MR, mitral regurgitation.\n\nTEE is mandatory in the operating room to confirm optimal competence of the valve after repair. Moreover, it is essential to confirm anatomical eligibility for percutaneous EE repair, where the anatomical criteria of the EVEREST II trial (Endovascular Valve Edge-to-Edge Repair Study) are the reference (Figure 3). Percutaneous treatments beyond these criteria are now common, although certain anatomical conditions predict failure or suboptimal outcomes (Figure 3).\n\nEVEREST II, Endovascular Valve Edge-to-Edge Repair Study; MR, mitral regurgitation.\n\n\nDegenerative mitral regurgitation\n\nThe natural history of severe degenerative MR is unfavorable and symptoms, age, AF, pulmonary hypertension, left atrial or LV dilatation, and low EF are all predictors of poor outcome5,6. There is no evidence-based medical therapy for patients with primary MR and minimal or no symptoms. Although beta-blockers and angiotensin-converting enzyme inhibitors palliate symptoms in patients with heart failure, they should not postpone the timing of intervention6. Although patients with primary MR could remain asymptomatic for years, thus deferring the intervention, the treatment strategy has been redefined in recent years. Nowadays the international guidelines recommend earlier intervention when the probability of durable repair is high, especially when surgery can be performed by skilled teams with excellent outcomes. The purpose of ‘early repair’ is to treat severe degenerative MR before the occurrence of structural and functional changes in the left atrial and LV chambers, to ensure that survival and quality of life of patients are similar to those of the general population.\n\nMV repair is the preferred surgical treatment for severe degenerative MR and has significant advantages over MV replacement6,11. The main goals (restitution of physiological leaflet motion, achievement of adequate leaflet coaptation, and annular stabilization with maintenance of an adequate mitral orifice) can be achieved by using a variety of isolated or combined techniques (leaflet resection, implantation of artificial chordae, chordal transposition/transfer, EE technique, and annuloplasty using a prosthetic ring or band) according to the type and location of the mitral lesions. Nowadays the vast majority of degenerative MR can be successfully repaired in dedicated valve centers12,13. Recent reports assess an absence from re-intervention at 10 years of more than 90%, slightly decreasing (to 80%) at 20 years14–16. Numerous anatomical lesions limiting long-term outcomes, especially the anterior or bileaflet prolapse, the extensive myxomatous disease, and annular calcifications, have been recognized so far. Surgical outcomes are strongly related to the experience of the center and surgeons. The hospital mortality achieved in dedicated centers is very low (less than 1%), and major complications are very rare when a strategy of early repair is adopted. Patients should be referred to centers with extensive experience to maximize the likelihood of a durable repair17,18. Indeed, the long-term outcomes are significantly improved when timely MV repair is performed, as opposed to lower late survival obtained in patients with hemodynamic decompensation (heart failure, depressed ventricular function, pulmonary hypertension, and arrhythmias)15,19.\n\nSeveral new transcatheter mitral devices are currently under investigation, although the MitraClip System (Abbott Vascular, Santa Clara, CA, USA), approved for use in high-risk or inoperable patients with severe MR and suitable anatomic criteria (Figure 3), is the only one widely available, and around 30,000 implantations are performed worldwide20. Percutaneous EE repair with this device is safe in degenerative MR and has low rates of procedural and 30-day mortality, complications (stroke, bleeding, tamponade, or resuscitation), and short mean hospital stay21–23.One-year survival is 80%, mirroring the advanced age and multiple comorbidities of the populations studied. Post-procedural mitral stenosis is very rare, and the clip detachment rate is around 2%. The acute procedural success rate (moderate or less than moderate final MR grade) is more than 80–85% and is maintained at 1- and 4-year follow-up. In the EVEREST II study21,24, which compared MV surgery versus transcatheter EE, 279 patients with moderate-to-severe and severe MR were randomly assigned to MitraClip repair (Abbott Laboratories, Abbott Park, IL, USA) or surgery (repair or replacement). The great majority of the population had degenerative MR and low risk of intervention. After 1 year, percutaneous repair was associated with a higher rate of residual MR, requiring surgical correction in comparison with surgery (20% versus 2%). These results were confirmed and remained stable after 4 years (25% versus 5%). Importantly, improvements in safety in favor of the percutaneous technique were influenced by the higher need for blood transfusion in the surgical arm. This population of the EVEREST II was significantly different from the patients who are currently treated in Europe, mainly affected by functional MR with severe LV dysfunction and higher surgical risk due to higher comorbidities. However, our group clearly documented that residual moderate MR after MitraClip implantation was associated with worse follow-up outcomes compared with mild or trivial MR, including survival, symptom relief, and MR recurrence. So far, better efficacy should be pursued by transcatheter mitral repair technologies in this particular setting25. Accordingly, the study by Suri et al. demonstrated that also recurrent MR following surgical mitral repair in degenerative patients is associated with adverse LV remodeling and late death26.\n\n\nFunctional mitral regurgitation\n\nMedical therapy (angiotensin-converting enzyme inhibitors, beta-blockers, and aldosterone antagonists) is mandatory in functional MR. Diuretics may be required for fluid overload, and vasodilators have a role in acute hemodynamic decompensation. The presence of conduction disturbances is not uncommon in these patients and contributes to the worsening of symptoms due to asynchronous ventricular contraction. Hence, cardiac resynchronization therapy aims at three different levels: (1) atrioventricular (2) intraventricular, and (3) interventricular. The therapy is achieved by pacing or sensing the right atrium, pacing the right ventricle (close to the septum), and pacing the left ventricle through the coronary sinus, also called biventricular pacing27.\n\nThe best surgical treatment for secondary MR remains controversial27. Mitral repair performed with an undersized rigid complete ring to restore leaflet coaptation and valve competence has been considered the standard treatment and can be performed with acceptable perioperative risk in carefully selected patients with secondary MR and poor LV function28,29. Several predictors of failure after repair have been recognized in the last decade, and it is well known that more advanced leaflet tethering predicts significant recurrent MR30–37. To improve MV repair durability, concomitant techniques on the subvalvular apparatus (secondary chordal resection, suturing of the posteromedial papillary muscle to the aorto-mitral continuity, infarct plication, papillary muscle imbrication, and posterior LV restoration) have been described in small, non-randomized, and observational studies and are under investigation30–37. Restrictive annuloplasty was recently compared with chordal-sparing MV replacement in a randomized study in patients with secondary MR of ischemic origin and demonstrated no advantage with regard to LV end-systolic volume index or 1-year mortality38. However, the trial was underpowered for mortality and included patients with pre-operative predictors of repair failure in the repair group. A more appropriate selection of the candidates to mitral repair should be pursued since the rates of moderate-to-severe recurrent MR at 1 year were 32.6% in the repair and 2.3% in the replacement group. When the study follow-up was extended to 2 years, no significant difference between groups in LV reverse remodeling or survival was documented, and values of moderate-to-severe recurrent MR were 58.8% in the repair group and 3.8% in the replacement39. Since no reverse LV remodeling was observed in the patients with recurrent MR, such a high rate of repair failure certainly had a major impact on the results. When repair was successful (no MR recurrence), the degree of LV reverse remodeling was higher than in patients submitted to MV replacement, emphasizing that a successful repair outplays the best MV replacement in this setting39. Therefore, further studies are required to determine whether selected patients with secondary MR and no predictors of repair failure may benefit from surgical MV reconstruction. Moreover, no study has convincingly demonstrated a survival benefit compared with medical therapy in patients with MR and LV systolic dysfunction and this argues against surgical intervention in asymptomatic patients and poses a complex surgical decision in high-risk cases40. However, in a large retrospective study recently published by Duke Medicine, substantial mortality was shown in patients with severe LV dysfunction and significant MR when treated with medical therapy alone whereas MV surgery was independently associated with higher event-free survival, encouraging the treatment of moderate and severe secondary MR in these challenging patients41.\n\nNowadays inoperable functional MR is widely treated with the MitraClip system, the transcatheter technology adopting the EE repair22,23,42–44. The outcomes of patients with functional MR and severe LV dysfunction in the ACCESS-EU (a prospective, multicenter, nonrandomized post-approval observational study of the MitraClip System in Europe) registry (around 400 patients) showed an extremely low mortality after 1 month (3%), which increased after 1 year to 17%, without evidence of significant complications (stroke, resuscitation, and tamponade)22,23. Significant residual MR in this challenging population, defined as moderate-to-severe or severe MR, progressively increased during the first year and was present in more than 20% of patients. With regard to clinical efficacy, most patients (70%) remained in New York Heart Association (NYHA) class I and II after the first year, showing either atrial or ventricular reverse remodeling, although in half of them a moderate residual MR was detected42–46. Direct comparisons between percutaneous EE repair and surgery in secondary MR are difficult since patients who receive either strategy are significantly different. Taramasso et al. reported in a non-randomized series higher efficacy of surgery compared with percutaneous intervention (freedom from moderate-to-severe and severe MR at 1 year was 94% versus 79%)43. In contrast, post hoc analysis of the EVEREST II trial demonstrated equivalence of the two strategies in this setting24. However, in the absence of a medical therapy control group, it is not possible to establish whether either treatment has a positive impact on survival; ongoing randomized studies will address this question. Currently, three large randomized trials will help clarify the future role of transcatheter devices in secondary MR therapy and whether MR reduction improves long-term outcomes: COAPT (Cardiovascular Outcomes Assessment of the MitraClip Percutaneous Therapy for Heart Failure Patients with Functional Mitral Regurgitation), RESHAPE-HF (Randomized Study of the MitraClip Device in Heart Failure Patients With Clinically Significant Functional Mitral Regurgitation), and MITRA-FR (Multicentre Study of Percutaneous Mitral Valve Repair MitraClip Device in Patients With Severe Secondary Mitral Regurgitation). The primary outcomes of these trials should be available at the end of 2017. In the COAPT trial (USA), 430 inoperable patients with secondary MR are randomly assigned between standard-of-care medical therapy and MitraClip versus standard-of-care medical therapy alone in order to assess the safety and effectiveness of the MitraClip in this field. In addition, the feasibility and safety of percutaneous direct mitral annuloplasty with Cardioband (Cardioband System; Valtech Cardio Ltd., Or Yehuda, Israel) have been recently assessed both in preclinical models and in humans47,48. The Cardioband system is a direct annuloplasty adjustable device that is implanted in the beating heart on the posterior annulus under fluoroscopic and TEE guidance. The human study group included 31 consecutive high-risk patients with moderate-to-severe or severe secondary MR48. Procedural mortality was zero, and in-hospital mortality was 6.5% (2 of 31 patients, neither procedure- nor device-related). At 1 month, 88% of patients had moderate or less than moderate residual MR.\n\nAlthough this work aims to report the current management of MV repair, it is worth mentioning the current role of the complementary therapy: the transcatheter valve replacement. The feasibility of transcatheter MV replacement has been reported in a small number of patients at extreme risk (fewer than 100 patients) with native, MV disease but does not allow for any robust conclusions. On one hand, implantation of a valve in a non-calcified MV raises several important challenges: its positioning and anchoring, causing obstruction of the LV outflow tract, or coronary circumflex artery or paravalvular leak. On the other hand, transcatheter MV replacement has several theoretical advantages (compared with valve repair) because it is versatile and durably eliminates MR49. Of the 10 ongoing studies, four are early feasibility trials in the US: Neovasc Tiara Mitral Valve System (TIARA-I; NCT02276547), Tendyne Mitral Valve System (NCT02321514), CardiAQ TMVI System (Transfemoral and Transapical DS; NCT02515539), and Twelve Transcatheter Mitral Valve Replacement (NCT02428010).\n\n\nConclusions\n\nNowadays surgical MV repair is considered the gold standard for patients with severe degenerative MR. To ensure the best durable outcomes, the procedure should be performed in a timely manner and in dedicated centers. In patients with secondary MR and dilated cardiomyopathy, the mitral repair intervention is more challenging and the careful selection of patients is essential. The presence of echocardiographic predictors of postoperative residual or recurrent MR should be carefully considered to recommend replacement as a more durable solution. Percutaneous interventions offer beating-heart MV repair and replacement under physiological conditions, without the need for cardiopulmonary bypass. Beyond percutaneous EE repair, the percutaneous direct annuloplasty reproduces proven surgical techniques, showing a good safety profile and efficacy. Advanced imaging technologies (three-dimensional echocardiography and heart computed tomography scan) will guide MV repair procedures in the near future.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nNkomo VT, Gardin JM, Skelton TN, et al.: Burden of valvular heart diseases: a population-based study. Lancet. 2006; 368(9540): 1005–11. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nIung B, Vahanian A: Epidemiology of valvular heart disease in the adult. Nat Rev Cardiol. 2011; 8(3): 162–72. PubMed Abstract | Publisher Full Text\n\nBursi F, Enriquez-Sarano M, Nkomo VT, et al.: Heart failure and death after myocardial infarction in the community: the emerging role of mitral regurgitation. Circulation. 2005; 111(3): 295–301. PubMed Abstract | Publisher Full Text\n\nGrigioni F, Enriquez-Sarano M, Zehr KJ, et al.: Ischemic mitral regurgitation: long-term outcome and prognostic implications with quantitative Doppler assessment. Circulation. 2001; 103(13): 1759–64. PubMed Abstract | Publisher Full Text\n\nLing LH, Enriquez-Sarano M, Seward JB, et al.: Clinical outcome of mitral regurgitation due to flail leaflet. N Engl J Med. 1996; 335(19): 1417–23. PubMed Abstract | Publisher Full Text\n\nJoint Task Force on the Management of Valvular Heart Disease of the European Society of Cardiology (ESC), European Association for Cardio-Thoracic Surgery (EACTS), Vahanian A, et al.: Guidelines on the management of valvular heart disease (version 2012). Eur Heart J. 2012; 33(19): 2451–96. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nStone GW, Vahanian AS, Adams DH, et al.: Clinical trial design principles and endpoint definitions for transcatheter mitral valve repair and replacement: part 1: clinical trial design principles: A consensus document from the mitral valve academic research consortium. Eur Heart J. 2015; 36(29): 1851–77. 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Circulation. 2001; 104(12 Suppl 1): I8–11. PubMed Abstract | Publisher Full Text\n\nDavid TE, Armstrong S, McCrindle BW, et al.: Late outcomes of mitral valve repair for mitral regurgitation due to degenerative disease. Circulation. 2013; 127(14): 1485–92. PubMed Abstract | Publisher Full Text\n\nDiBardino DJ, ElBardissi AW, McClure RS, et al.: Four decades of experience with mitral valve repair: analysis of differential indications, technical evolution, and long-term outcome. J Thorac Cardiovasc Surg. 2010; 139(1): 76–83; discussion 83–4. PubMed Abstract | Publisher Full Text\n\nIung B, Baron G, Butchart EG, et al.: A prospective survey of patients with valvular heart disease in Europe: The Euro Heart Survey on Valvular Heart Disease. Eur Heart J. 2003; 24(13): 1231–43. PubMed Abstract | Publisher Full Text\n\nAnyanwu AC, Bridgewater B, Adams DH: The lottery of mitral valve repair surgery. Heart. 2010; 96(24): 1964–7. PubMed Abstract | Publisher Full Text\n\nMontant P, Chenot F, Robert A, et al.: Long-term survival in asymptomatic patients with severe degenerative mitral regurgitation: a propensity score-based comparison between an early surgical strategy and a conservative treatment approach. J Thorac Cardiovasc Surg. 2009; 138(6): 1339–48. PubMed Abstract | Publisher Full Text\n\nMinha S, Torguson R, Waksman R: Overview of the 2013 Food and Drug Administration Circulatory System Devices Panel meeting on the MitraClip Delivery System. Circulation. 2013; 128(8): 864–8. PubMed Abstract | Publisher Full Text\n\nFeldman T, Foster E, Glower DD, et al.: Percutaneous repair or surgery for mitral regurgitation. N Engl J Med. 2011; 364(15): 1395–406. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMaisano F, Franzen O, Baldus S, et al.: Percutaneous mitral valve interventions in the real world: early and 1-year results from the ACCESS-EU, a prospective, multicenter, nonrandomized post-approval study of the MitraClip therapy in Europe. J Am Coll Cardiol. 2013; 62(12): 1052–61. PubMed Abstract | Publisher Full Text\n\nReichenspurner H, Schillinger W, Baldus S, et al.: Clinical outcomes through 12 months in patients with degenerative mitral regurgitation treated with the MitraClip® device in the ACCESS-EUrope Phase I trial. Eur J Cardiothorac Surg. 2013; 44(4): e280–8. PubMed Abstract | Publisher Full Text\n\nMauri L, Foster E, Glower DD, et al.: 4-year results of a randomized controlled trial of percutaneous repair versus surgery for mitral regurgitation. J Am Coll Cardiol. 2013; 62(4): 317–28. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBuzzatti N, De Bonis M, Denti P, et al.: What is a \"good\" result after transcatheter mitral repair? Impact of 2+ residual mitral regurgitation. J Thorac Cardiovasc Surg. 2016; 151(1): 88–96. PubMed Abstract | Publisher Full Text\n\nSuri RM, Clavel MA, Schaff HV, et al.: Effect of Recurrent Mitral Regurgitation Following Degenerative Mitral Valve Repair: Long-Term Analysis of Competing Outcomes. J Am Coll Cardiol. 2016; 67(5): 488–98. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMcMurray JJ, Adamopoulos S, Anker SD, et al.: ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC. Eur Heart J. 2012; 33(14): 1787–847. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMagne J, Girerd N, Sénéchal M, et al.: Mitral repair versus replacement for ischemic mitral regurgitation: comparison of short-term and long-term survival. Circulation. 2009; 120(11 Suppl): S104–11. PubMed Abstract | Publisher Full Text\n\nBolling SF, Deeb GM, Brunsting LA, et al.: Early outcome of mitral valve reconstruction in patients with end-stage cardiomyopathy. J Thorac Cardiovasc Surg. 1995; 109(4): 676–82; discussion 682–3. PubMed Abstract | Publisher Full Text\n\nMcGee EC Jr, Gillinov AM, Blackstone EH, et al.: Recurrent mitral regurgitation after annuloplasty for functional ischemic mitral regurgitation. J Thorac Cardiovasc Surg. 2004; 128(6): 916–24. PubMed Abstract | Publisher Full Text\n\nCiarka A, Braun J, Delgado V, et al.: Predictors of mitral regurgitation recurrence in patients with heart failure undergoing mitral valve annuloplasty. Am J Cardiol. 2010; 106(3): 395–401. PubMed Abstract | Publisher Full Text\n\nLee AP, Acker M, Kubo SH, et al.: Mechanisms of recurrent functional mitral regurgitation after mitral valve repair in nonischemic dilated cardiomyopathy: importance of distal anterior leaflet tethering. Circulation. 2009; 119(19): 2606–14. PubMed Abstract | Publisher Full Text\n\nKron IL, Hung J, Overbey JR, et al.: Predicting recurrent mitral regurgitation after mitral valve repair for severe ischemic mitral regurgitation. J Thorac Cardiovasc Surg. 2015; 149(3): 752–61.e1. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBorger MA, Alam A, Murphy PM, et al.: Chronic ischemic mitral regurgitation: repair, replace or rethink? Ann Thorac Surg. 2006; 81(3): 1153–61. PubMed Abstract | Publisher Full Text\n\nBorger MA, Murphy PM, Alam A, et al.: Initial results of the chordal-cutting operation for ischemic mitral regurgitation. J Thorac Cardiovasc Surg. 2007; 133(6): 1483–92. PubMed Abstract | Publisher Full Text\n\nLanger F, Kunihara T, Hell K, et al.: RING+STRING: Successful repair technique for ischemic mitral regurgitation with severe leaflet tethering. Circulation. 2009; 120(11 Suppl): S85–91. PubMed Abstract | Publisher Full Text\n\nHvass U, Joudinaud T: The papillary muscle sling for ischemic mitral regurgitation. J Thorac Cardiovasc Surg. 2010; 139(2): 418–23. PubMed Abstract | Publisher Full Text\n\nAcker MA, Parides MK, Perrault LP, et al.: Mitral-valve repair versus replacement for severe ischemic mitral regurgitation. N Engl J Med. 2014; 370(1): 23–32. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGoldstein D, Moskowitz AJ, Gelijns AC, et al.: Two-Year Outcomes of Surgical Treatment of Severe Ischemic Mitral Regurgitation. N Engl J Med. 2016; 374(4): 344–53. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWu AH, Aaronson KD, Bolling SF, et al.: Impact of mitral valve annuloplasty on mortality risk in patients with mitral regurgitation and left ventricular systolic dysfunction. J Am Coll Cardiol. 2005; 45(3): 381–7. PubMed Abstract | Publisher Full Text\n\nSamad Z, Shaw LK, Phelan M, et al.: Management and outcomes in patients with moderate or severe functional mitral regurgitation and severe left ventricular dysfunction. Eur Heart J. 2015; 36(40): 2733–41. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSchillinger W, Hünlich M, Baldus S, et al.: Acute outcomes after MitraClip therapy in highly aged patients: results from the German TRAnscatheter Mitral valve Interventions (TRAMI) Registry. EuroIntervention. 2013; 9(1): 84–90. PubMed Abstract | Publisher Full Text\n\nTaramasso M, Maisano F, Latib A, et al.: Clinical outcomes of MitraClip for the treatment of functional mitral regurgitation. EuroIntervention. 2014; 10(6): 746–52. PubMed Abstract | Publisher Full Text\n\nGrayburn PA, Foster E, Sangli C, et al.: Relationship between the magnitude of reduction in mitral regurgitation severity and left ventricular and left atrial reverse remodeling after MitraClip therapy. Circulation. 2013; 128(15): 1667–74. PubMed Abstract | Publisher Full Text\n\nGrasso C, Capodanno D, Scandura S, et al.: One- and twelve-month safety and efficacy outcomes of patients undergoing edge-to-edge percutaneous mitral valve repair (from the GRASP Registry). Am J Cardiol. 2013; 111(10): 1482–7. PubMed Abstract | Publisher Full Text\n\nVan den Branden BJ, Swaans MJ, Post MC, et al.: Percutaneous edge-to-edge mitral valve repair in high-surgical-risk patients: do we hit the target? JACC Cardiovasc Interv. 2012; 5(1): 105–11. PubMed Abstract | Publisher Full Text\n\nTaramasso M, Guidotti A, Cesarovic N, et al.: Transcatheter direct mitral annuloplasty with Cardioband: feasibility and efficacy trial in an acute preclinical model. EuroIntervention. 2015; 11(7): pii: EIJ-D-15-00021. PubMed Abstract | Faculty Opinions Recommendation\n\nMaisano F, Taramasso M, Nickenig G, et al.: Cardioband, a transcatheter surgical-like direct mitral valve annuloplasty system: early results of the feasibility trial. Eur Heart J. 2016; 37(10): 817–25. PubMed Abstract | Publisher Full Text\n\nMaisano F, Alfieri O, Banai S, et al.: The future of transcatheter mitral valve interventions: competitive or complementary role of repair vs. replacement? Eur Heart J. 2015; 36(26): 1651–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "14295",
"date": "10 Jun 2016",
"name": "Pieter Kappetein",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14296",
"date": "10 Jun 2016",
"name": "Javier Castillo",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14297",
"date": "10 Jun 2016",
"name": "Nicolas Doll",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1326
|
https://f1000research.com/articles/5-1313/v1
|
09 Jun 16
|
{
"type": "Research Note",
"title": "Challenges in implementing the 2015 BASHH guidelines for the appropriate use of post-exposure prophylaxis for HIV following sexual exposure",
"authors": [
"Mohammad Fawad Khattak"
],
"abstract": "The use of post exposure prophylaxis for human immunodeficiency virus (HIV) following sexual exposure (PEPSE) was retrospectively audited in an inner city genitourinary clinic against the 2015 national guidelines by the British Association for Sexual Health and HIV (BASHH). One-hundred out of a total of 101 patients (99%) had a baseline HIV test done. 82.1% (n=83) of patients were given PEPSE prescriptions fitting within recommended indications lower than the 90% target set by BASHH. 84.2% (n=85) of patients had PEPSE administered within 72 hours lower than the 90%. 61.4% (n=62) of patients were known to have completed four weeks of PEPSE lower than the 75% target. 61.4% (n=62) of patients were screened for sexually transmitted infections (STIs) lower than the 90% target. 59.4% (n=60%) of patients had post-PEPSE HIV bloods slightly lower than the 60% target.",
"keywords": [
"HIV",
"post exposure prophylaxis for HIV",
"BASHH",
"antiretrovirals",
"sexually transmitted infections"
],
"content": "Introduction\n\nPost-exposure prophylaxis for HIV involves taking antiretrovirals by human immunodeficiency virus (HIV)-negative individuals for four weeks, after a suspected or known exposure to HIV to reduce the risk of transmission1,2. In 2015 the British Association for Sexual Health and HIV updated national guidelines on the appropriate use of post exposure prophylaxis after sexual exposure to HIV (PEPSE)3. -- The guidelines provide indications for when PEPSE use; is recommended; can be considered; or is not appropriate. It also recommends PEPSE use within 72 hours; baseline HIV testing, appropriate sexually transmitted infection (STI) testing, and completion of four weeks of PEPSE with follow up HIV bloods after completion of PEPSE. BASHH have specified auditable targets for these recommendations, and this retrospective audit compares the use of PEPSE in our genitourinary clinic against these recommendations.\n\n\nMethod\n\nA retrospective case note review was carried out at Walsall Centre of Sexual Health. One-hundred one patients who were coded as having received PEPSE between June 2013 and September 2015 were identified on the computer system. No permission was required to conduct the study and publish these results. Notes of these patients were reviewed and data regarding; the indication of PEPSE administration; time since exposure; investigations carried out; completion of four weeks of PEPSE and whether the patient had follow up investigation were uploaded onto a Microsoft Excel database.\n\n\nResults\n\nThe results of 101 patients who received PEPSE were analysed (Table 1). 48.5% (n=49) of patients were male, 61% (n=30) of the male patients were bi-sexual/homosexual.\n\nBaseline HIV tests were done in 99% of patients (n=100). One patient did not have baseline HIV tests. This patient initially visited a local emergency department where she received PEPSE without HIV testing. This patient subsequently came to our genitourinary medicine clinic one week later and had a HIV test done. Baseline HIV test was done on the first visit to the clinic in all 100 cases.\n\n52.5% (n=53/101) of prescriptions for PEPSE were given under recommended indications by BASHH (Table 2), and 29.7% (n=30/101) of patients were given PEPSE under indications where BASHH state they can be considered. All of the patients in the considered category in this audit were female patients who had been sexually abused. In total, 82.2% of patients were given PEPSE for recommended/considered indications lower than the 90% target. 5% (n=5/101) of patients had no documented reason for starting PEPSE.\n\n84.2% (n=85) of patients received PEPSE within 72 hours of exposure lower than the 90% target. 13.9% (n=14) of PEPSE was prescribed after 72 hours since exposure, while 5% (n=5) of patients had no documentation of when the exposure occurred.\n\n61.4% (n=62) of patients had documentation showing that they had completed four weeks of PEPSE, lower than the 75% target. 13.9% (n=14) of patients did not complete four weeks of PEPSE while 24.8% (n=25) of patients had no documentation regarding whether they had completed four weeks of PEPSE.\n\n59.4% (n=60%) of patients had post-PEPSE HIV bloods slightly lower than the 60% target.\n\nAll 100 patients that had baseline HIV bloods taken had appropriate investigations into hepatitis B and syphilis. However 61.4% (n=62) of patients were screened for chlamydia and gonorrhoea lower than the 90% target. None of the patients who had come for PEPSE after needle stick injury had testing for gonorrhoea and chlamydia, and not taking into account these patients 73% of patients had screening for chlamydia and gonorrhoea.\n\n\nDiscussion\n\nThe majority of patients prescribed PEPSE where given so under indications that were deemed compliant with BASHH guidelines and within 72 hours of the suspected exposure. All but one of these patients had baselines HIV bloods taken, and appropriate testing for syphilis and hepatitis B, with post-PEPSE follow-up testing levels being near the BASHH target.\n\nDocumentation regarding whether patients were taking or discontinuing PEPSE was lacking. There was also difficulty determining whether patients who did not attend after their initial visit had completed their PEPSE course.\n\nOne particular guideline that we found difficulty in reaching was screening for chlamydia and gonorrhoea, especially in patients coming in after needlestick injuries. Often times it is either not considered suitable or the patient declines the screening as they do not feel they are at risk. Screening should always be encouraged and there should be documentation that the screening tests have been declined if that is case.\n\nAnother issue is patients not visiting for follow-ups. Often screening for chlamydia and gonorrhoea is delayed until after the window period for investigations to identify these organisms. Follow-ups are also important for identifying whether the patient is compliant with the antiretrovirals, and for post-PEPSE HIV blood tests.\n\nCarrying out an audit against the BASHH guidelines have highlighted areas in our clinical practice which need improvement. In response to this audit we have created clear proformas for prescribing PEPSE which include; whether the indication the patient is coming in with fits with BASHH guidelines; whether the exposure was less than 72 hours ago, and a list of the relevant investigations that should be considered. Proformas for follow-up have also been made to assess whether the patient is completing the course of PEPSE and having follow-up bloods after completion of therapy. Training has been given to educate all staff on the indications for PEPSE prescribing, the need to identify HIV status and viral load of source, the need to have accurate documentation, to offer rapid-HIV testing, and fourth generation HIV testing for post-PEPSE follow-up. We have also decided to get patients to book their follow-up appointment during their initial visit to the clinic. We will then send SMS reminders the day before the follow-up appointment to remind patients to attend. We plan to carry out a re-audit in one year.\n\n\nData availability\n\nAll raw data are provided in the tables above.",
"appendix": "Competing interests\n\n\n\nThe authors declare no conflict of interest.\n\n\nGrant information\n\nThe author declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPinto LA, Landay AL, Berzofsky JA, et al.: Immune response to human immunodeficiency virus (HIV) in healthcare workers occupationally exposed to HIV-contaminated blood. Am J Med. 1997; 102(5B): 21–24. PubMed Abstract | Publisher Full Text\n\nBourry O, Mannioui A, Sellier P, et al.: Effect of a short-term HAART on SIV load in macaque tissues is dependent on time of initiation and antiviral diffusion. Retrovirology. 2010; 7: 78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBritish Association for Sexual Health and HIV: United Kingdom guideline for the use of post-exposure prophylaxis for HIV following sexual exposure. 2015; accessed 19 November 2015. Reference Source"
}
|
[
{
"id": "22009",
"date": "20 Apr 2017",
"name": "Theresa Marié Rossouw",
"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 are some language and grammatical errors that need attention.\n\nThe link provided to the BASHH guidelines is for the draft document and not the final version.\n\nMore detail about the context is need e.g. where is the Walsall Centre of Sexual Health located?\n\nReference 1: I could unfortunately not access the full article, but the reference to this 1997 article in support of post-exposure prophylaxis does not seem to be the best choice since it deals with \"Immune response to human immunodeficiency virus (HIV) in health care workers occupationally exposed to HIV-contaminated blood\" and does not seem to deal with post-exposure prophylaxis.\n\nThe second reference in support of PEP is also not the most relevant reference since it is an animal study and the final conclusion of this article is that “limited antiviral drug diffusion in secondary lymphoid tissues may allow persistent viral replication in these tissues and could represent an obstacle to HIV prevention and eradication”.\n\nIt would be interesting to know how many of the patients were repeat users of PEPSE. A possible recommendation could be monitoring for repeat users of PEPSE and referring them for PrEP.\n\nIt would also be interesting to know what antiretroviral medication had been prescribed for PEPSE and whether this complied with the guidelines.\n\nIt is preferable not to refer to \"bi-sexual/homosexual\" but rather to men who have sex with men and/or women.\n\nTable 1: Under “Baselines HIV Test” and “Post-PEPSE HIV Test”, grammar and placing of results should receive attention.\n\nTable 2 needs more explanation since the numbers do not add up. For instance, the text shows that 53/101 had a recommendation for PEPSE, whereas the table shows only 20 HIV+ with unknown or high VL and 18 MSM. The text shows that 30/101 had circumstances under which PEPSE could be considered but the table shows 31 cases of sexual assault. There were 15 cases of needle stick injury. This seems to be very high and since the guideline only covers needle stick injuries in the community, the circumstances surrounding the injuries should be explained. I recommend that all exposure categories be listed in order to improve understanding. It seems that Tables 1 and 2 could be merged.\n\nTable 2: The recommended VL above which PEPSE should be given is 200 copies/ml and not 40 copies/ml – the unit of measure is not given in the table. In addition, the meaning of the category “Men who have sex with men (MSM) High prevalence/unknown” is unclear.\n\nThe proportion of PEPSE prescriptions administered within 24 hours (and not 72 hours) of risk exposure is the auditable outcome and should also be shown.\n\nThe author states that “There was also difficulty determining whether patients who did not attend after their initial visit had completed their PEPSE course”. What measures were taken to determine this?\n\nWhat steps had been taken to assess the VL of HIV-infected partners?\n\n“No permission was required to conduct the study and publish these results.” I presume the author means that informed consent was not considered relevant since the data used were anonymous, but surely institutional permission was needed?\n\nArticle should make reference to other related work, e.g. Ladipo Z, Chauhan M, Foster K. Survey of PEPSE provision: lessons to be learnt. Sex Transm Infect 2015;91:1641.\n\nCurrie S, Shafiq V, Mellor J, et al. P73 The source of the problem – re-audit of pepse provision at an inner city sexual health clinic. Sex Transm Infect 2015;91:A39-A402\n\nRaha D; Gilson R. An audit of PEPSE in an integrated sexual and reproductive health centre resulting in change of local protocol in accordance to national guidelines: room for improvement. HIV Medicine. 17():33–34, Apr 20163\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "22154",
"date": "26 May 2017",
"name": "Paul Volberding",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting report of the use of post-exposure prophylaxis following possible sexual exposure to HIV in a UK retrospective cohort. While it shows that British guidelines were generally followed well, it of course leaves open the question of what effect the prophylaxis had in terms of eventual HIV seroconversion. Another question raised is how many of those receiving PEP in this study may have also been appropriate candidates for PeEP given our current knowledge of the effectiveness of that prevention approach.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "23142",
"date": "07 Jun 2017",
"name": "Rachel Beanland",
"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 of a clinical audit conducted to assess the implementation of UK BASHH guidelines on Post Exposure Prophylaxis following sexual exposure. The paper provides a clear summary of the methods and results compared to national guidelines. The recommendations and subsequent actions taken following the results are of interest to the audience. Further clarification in some sections could enhance the key message of the paper. In places the grammar and language could be improved.\n\nComments and considerations for the authors:\n\nMethod\n\nPlease provide further geographical detail (region, country) to allow the audience to understand the context of the setting. Consider rewording the sentence regarding permission. As secondary data has been used no patient consent was needed but how was confidentiality maintained, was the data anonymised? Did you seek any ethical approval or institutional approval? Note Excel is not a classic database, please revise to spreadsheet, unless alternative database was used for analysis.\n\nResults\n\nConsider using 'performed' or 'conducted' in place of 'done'. Table 2. The article focuses on prophylaxis from sexual exposure, it is not clear why needle stick injury is included in this data set. If this is the case, please specify why included. Please review the data presented in the table for accuracy. Do you have any detail on the ARVs prescribed and how adherent the individuals were?\n\nReferences Please review the references and provide uptodate citations. WHO PEP guidelines http://apps.who.int/iris/bitstream/10665/145719/1/9789241508193_eng.pdf the supporting evidence Clinical Infectious Disease supplement https://academic.oup.com/cid/issue/60/suppl_3 be helpful.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1313
|
https://f1000research.com/articles/5-1311/v1
|
09 Jun 16
|
{
"type": "Research Note",
"title": "Facebook and mental wellbeing: a crossover randomised controlled study",
"authors": [
"Achim Wolf"
],
"abstract": "Objective: To study the effect of reducing Facebook use for two weeks on self-reported mental wellbeing in a student population. Methods: Students at the University of Oxford (n=78) participated in a randomised crossover study which consisted of two consecutive two-week periods of minimised Facebook use, followed by normal Facebook use, or vice-versa. Participants were evaluated after each two-week period using the 14-item Warwick-Edinburgh Mental Well-Being Scale (WEMWBS), and completed questionnaires about their Facebook use. Differences in WEMWBS scores were evaluated using a paired t-test. Results: Of those enrolled, 57 (73%) students completed the entire study. 93% reported reduced Facebook use during the intervention period. When limiting their Facebook use, participants had an average WEMWBS score of 46.0, compared to 43.7 during the control period, equating to a difference of 2.3 points (95% CI: 0.4 to 4.2; p=0.016; Cohen’s d = 0.33). There were no significant differences in dropout between the two groups (p=0.3), or differences in effect when stratifying by gender (p=0.9) or relationship status (p=0.6). Conclusion: Reducing Facebook use may be an effective intervention for improving mental wellbeing in university students. Future studies should examine effects in other participant groups, and use longer follow-up periods.",
"keywords": [
"Facebook",
"mental wellbeing",
"randomised controlled study",
"WEMWBS"
],
"content": "Introduction\n\nFacebook is the largest online social networking service with around 1.1 billion monthly active users1.\n\nPrevious work has come to different conclusions on the effects of Facebook on mental wellbeing2,3. Despite a growing body of research surrounding the effects and implications of Facebook’s popularity4, no research has used experimental methods to examine the Facebook and mental wellbeing relationship, and uncertainty around causality remains.\n\nTo address this, we have conducted an experimental study using a crossover randomised design. Students at the University of Oxford (n=78) were asked to limit their Facebook use for two weeks and complete questionnaires on their mental wellbeing.\n\n\nMethods\n\nThis study used a crossover design with simple randomisation (1:1 ratio), involving a two-week period with minimal Facebook use, and a two-week control period. No changes were made to the study protocol after receiving ethics approval through the University of Oxford’s Medical Sciences Interdivisional Research Ethics Committee (Ref#: MSD-IDREC-C1-2014-035), which was granted before commencement of the study. Trial registration was completed retrospectively through the Australian New Zealand Clinical Trials Registry (Ref#: ACTRN12614000959673, www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=367015).\n\nParticipants were recruited between March 27 and May 25, 2014 and had to be full-time students at the University of Oxford, aged over 18, and regular (daily) Facebook users. No exclusion criteria were used. Students were recruited through adverts in student newsletters and Facebook pages, and departmental mailing lists. They were followed up for four weeks, with follow-ups starting on March 27, 2014 and ending June 22, 2014. There was no face-to-face contact between participants and researchers, or compensation for participants.\n\nEligibility data, informed consent, and questionnaire answers were obtained electronically. To confirm eligibility, students had to register with a valid University of Oxford email address.\n\nAt the beginning of the intervention period, participants were asked to “restrict [their] Facebook use as much as possible for two weeks” and were told “During this time, please avoid using Facebook, including through mobile phones, tablets, and computers.” At the beginning of the control period, participants were told they “can use Facebook as much as [they] want” for two weeks. Participants were sent weekly reminders. Facebook use was measured through self-reporting only.\n\nAfter each two-week period participants were sent links with questionnaires, and were asked to complete them regardless of whether they had followed the instructions given to them.\n\nThe primary outcome is within-individual differences in Warwick-Edinburgh Mental Well-Being Scale (WEMWBS) score between the two periods. WEMWBS is a 14-item measure of positive mental wellbeing which has been validated in UK student populations5. Secondary outcomes were stratification by two variables: gender, and relationship status.\n\nThe sample size of 78 was calculated assuming a power of 0.8, an α of 0.05, a minimal detectable difference in means of 2 points in WEMWBS score, a 20% dropout rate, and a standard error of measurement (SEM) of 2.78 (mean of the five SEMs reported in Maheswaran et al.6).\n\nParticipants were randomised on a rolling basis after completing the informed consent form, using the rand() function in Excel 2013 (v.15.0). At randomisation, no data other than the participant’s email address had been collected. Participants were not blinded to group allocation.\n\nThe statistical significance of the primary outcome was calculated using a paired t-test. Only participants who completed both sets of questionnaires were entered in this analysis.\n\nIn a sensitivity analysis, all observations were entered into a regression analysis using Stata’s clustered sandwich estimator (vce(cluster) function) to allow for within-participant correlation in data with repeated observations on individuals7, and to minimise missingness.\n\nWe present self-reported descriptive statistics on age, gender, relationship status, and whether participants reduced their use during the intervention period.\n\nFor secondary outcomes, we stratified analyses by gender (female vs. male) and relationship status (single vs. in a relationship).\n\nAll analyses were done in Stata, version 12. Observations were entered according to group allocation, following an intention-to-treat approach. Statistical assumptions for paired t-tests were tested using a histogram of the differences in scores (Assumption of Normality, Supplementary figure 1) and by plotting differences in scores against average scores (Supplementary figure 2).\n\n\nResults\n\nAt randomisation, 40 participants were allocated to Group AB and 38 to Group BA. Two-thirds of participants were female, 46% were single. There was no significant difference in dropout between Group AB and Group BA (Table 1).\n\n1 Chi-square of difference in dropout (full vs. half or none) non-significant (p=0.3).\n\n2 Data shown for those with complete follow-up (both 2 and 4 weeks).\n\nDuring the intervention period 25/27 (93%) in Group AB and 28/30 (93%) in Group BA reported reduced Facebook usage (‘less than usual’ or ‘much less than usual’).\n\nParticipants obtained average WEMWBS scores of 46.0 (95% CI: 44.3 to 47.7) during the intervention period, and 43.7 (95% CI: 41.7 to 45.7) during the control period. The average difference was 2.3 points (95% CI: 0.4 to 4.2, p=0.016). This corresponds to a Cohen’s d of 0.33 (SD of difference = 7.0).\n\nIn a sensitivity analysis additionally including those with follow-up at 2 weeks only, the average difference in scores was 2.6 points (95% CI: 0.7 to 4.5, p=0.009).\n\nWe found no significant differences in effect by gender or relationship status (Table 2).\n\n\nDiscussion\n\nIn this crossover randomised study, we analysed the effects of reducing Facebook use on mental wellbeing. This is the first randomised study to look at the relationship between Facebook use and mental wellbeing. We find that participants had an average 2.3 point higher WEMWBS score after being asked to limit their Facebook use for two weeks. For comparison, a study examining a 12 week web-based cognitive behavioural tool to improve mental wellbeing in the general population found a 2.9 point difference using the same scale8. Our study supports the idea that associations reported in a previous study3 may in fact be causal.\n\nThe link between Facebook and mental wellbeing may be related to content perceived to be negative. A focus group found five main Facebook stressors: “managing inappropriate or annoying content, being tethered, lack of privacy and control, social comparison and jealousy, and relationship tension and conflict”9. In contrast, a study using Experience Sampling Methods found greater changes in self-esteem through text-based communications (including Facebook) than through face-to-face communication10. Differences in findings may be due to individual use patterns and feedback from other users11.\n\nLimitations include high dropout (possibly due to the lack of compensation) and a short follow-up period. Almost all students reported reduced Facebook use. Nevertheless, results could be affected by expectations, or perception of reduced use rather than actual changes in use.\n\n\nConclusion\n\nReducing Facebook use may be an effective intervention for improving mental wellbeing in university students. Future studies should examine effects in other participant groups, use longer follow-up periods, and consider deception to improve participant blinding.\n\n\nData availability\n\nAll collected data (excluding email addresses and names) are provided.\n\nF1000Research: Dataset 1. Raw data of facebook and mental wellbeing analysis, 10.5256/f1000research.8835.d12480612\n\n\nConsent\n\nWritten informed consent for publication of their details was obtained from all participants.",
"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\nSupplementary material\n\n\n\n\nReferences\n\nFacebook Inc: Facebook Reports Fourth Quarter and Full Year 2013 Results. 2014; Accessed 13/08/14. Reference Source\n\nValenzuela S, Park N, Kee KF: Is There Social Capital in a Social Network Site?: Facebook Use and College Students' Life Satisfaction, Trust, and Participation1. J Comput Mediat Commun. 2009; 14(4): 875–901. Publisher Full Text\n\nKross E, Verduyn P, Demiralp E, et al.: Facebook use predicts declines in subjective well-being in young adults. PLoS One. 2013; 8(8): e69841. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilson RE, Gosling SD, Graham LT: A Review of Facebook Research in the Social Sciences. Perspect Psychol Sci. 2012; 7(3): 203–20. PubMed Abstract | Publisher Full Text\n\nTennant R, Hiller L, Fishwick R, et al.: The Warwick-Edinburgh Mental Well-being Scale (WEMWBS): development and UK validation. Health Qual Life Outcomes. 2007; 5(1): 63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaheswaran H, Weich S, Powell J, et al.: Evaluating the responsiveness of the Warwick Edinburgh Mental Well-Being Scale (WEMWBS): group and individual level analysis. Health Qual Life Outcomes. 2012; 10(1): 156. PubMed Abstract | Publisher Full Text\n\nStataCorp L: Stata User’s Guide, Version 12. College Station, TX: Stata Press, StataCorp LP. 2011.\n\nPowell J, Hamborg T, Stallard N, et al.: Effectiveness of a web-based cognitive-behavioral tool to improve mental well-being in the general population: randomized controlled trial. J Med Internet Res. 2013; 15(1): e2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFox J, Moreland JJ: The dark side of social networking sites: An exploration of the relational and psychological stressors associated with Facebook use and affordances. Comput Hum Behav. 2015; 45: 168–76. Publisher Full Text\n\nGonzales AL: Text-based communication influences self-esteem more than face-to-face or cellphone communication. Comput Hum Behav. 2014; 39: 197–203. Publisher Full Text\n\nTobin SJ, Vanman EJ, Verreynne M, et al.: Threats to belonging on Facebook: lurking and ostracism. Soc Influ. 2015; 10(1): 31–42. Publisher Full Text\n\nWolf A: Dataset 1 in: Facebook and Mental Wellbeing: A crossover randomised controlled study. F1000Research. 2016. Data Source"
}
|
[
{
"id": "14288",
"date": "13 Jun 2016",
"name": "Shahadat Uddin",
"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 well designed and the author addressed the limitations and future research direction of the study very well. However, the manuscript lacks significantly in summarising previous research in align to this research. The author needs to describe a brief about the present status of literature in align to this study. Here is two examples of such literature:\nInterconnected or disconnected? Promotion of mental health and prevention of mental disorder in the digital age (Hayes, Maughan & Grant-Peterkin, 2016)1\nExploring physical, mental and psychological health for elders through their personal networks (Uddin & Hossain, 2009) 2",
"responses": []
},
{
"id": "17497",
"date": "09 Dec 2016",
"name": "Scottye J. Cash",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for asking me to review this manuscript. While the topic is interesting, the methods are limited, especially when considering the manuscript tried to make causal assertions. The methods were not very sophisticated and rather vague. Information on the sample was not included. There are many covariates (age, gender, year in school, length of time on Facebook, mental health history, etc.) that needed to be included in the analysis. The findings don't make a significant enough of an impact on the knowledge base.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1311
|
https://f1000research.com/articles/5-1309/v1
|
09 Jun 16
|
{
"type": "Software Tool Article",
"title": "RiboProfiling: a Bioconductor package for standard Ribo-seq pipeline processing",
"authors": [
"Alexandra Popa",
"Kevin Lebrigand",
"Agnes Paquet",
"Nicolas Nottet",
"Karine Robbe-Sermesant",
"Rainer Waldmann",
"Pascal Barbry",
"Alexandra Popa",
"Kevin Lebrigand",
"Agnes Paquet",
"Nicolas Nottet",
"Karine Robbe-Sermesant",
"Rainer Waldmann"
],
"abstract": "The ribosome profiling technique (Ribo-seq) allows the selective sequencing of translated RNA regions. Recently, the analysis of genomic sequences associated to Ribo-seq reads has been widely employed to assess their coding potential. These analyses led to the identification of differentially translated transcripts under different experimental conditions, and/or ribosome pausing on codon motifs. In the context of the ever-growing need for tools analyzing Ribo-seq reads, we have developed ‘RiboProfiling’, a new Bioconductor open-source package. ‘RiboProfiling’ provides a full pipeline to cover all key steps for the analysis of ribosome footprints. This pipeline has been implemented in a single R workflow. The package takes an alignment (BAM) file as input and performs ribosome footprint quantification at a transcript level. It also identifies footprint accumulation on particular amino acids or multi amino-acids motifs. Report summary graphs and data quantification are generated automatically. The package facilitates quality assessment and quantification of Ribo-seq experiments. Its implementation in Bioconductor enables the modeling and statistical analysis of its output through the vast choice of packages available in R. This article illustrates how to identify codon-motifs accumulating ribosome footprints, based on data from Escherichia coli.",
"keywords": [
"ribosome profiling",
"genomics",
"ribosome footprints"
],
"content": "Introduction\n\nRibosome profiling (Ribo-seq) is a recently developed high throughput sequencing technique (Ingolia et al., 2009) that allows the identification of RNA fragments resistant to RNAse digestion. Fragments mainly correspond to coding sequences protected against RNAse digestion by translating ribosomes. Ribo-seq data have been widely used to assess the translational status of open reading frames (ORFs) (Bazzini et al., 2014; Fields et al., 2015; Ingolia et al., 2009; Popa et al., 2016), and to identify ORFs differentially translated between experimental conditions (Schafer et al., 2015).\n\nRibo-seq bioinformatics analyses comprise the selection of reads consistent with ribosome footprints, a recalibration of the read start or end (5’ or 3’ extremity of the read depending on the RNA digestion step) to the peptidyl site (P-site) position of the ribosome, and quantification of the reads on specific features of interest (i.e. transcript, codons, multi-codons motifs). Several tools for processing ribosome profiling data have previously been proposed. RiboTools’ (Legendre et al., 2015) and ‘RUST’ (O’Connor et al., 2015)) were developed in python, ‘riboSeqR’ (Hardcastle, 2014) corresponds to an R package. Each of the above software integrates some, but not all, of the functions necessary for a standard Ribo-seq workflow from reads to quality assessment, recalibration and quantification. A previous effort to group the different approaches for Ribo-seq analyses has been developed with the Galaxy instance RiboGalaxy (Michel et al., 2016).\n\nThese tools have been developed to answer specific questions related to ribosome occupancy: normalization of Ribo-seq reads (‘RUST’), detection and characterization of reading frame usage (‘riboSeqR’), or irregular translational behavior such as translational ambiguities (Ribo-seq footprints in different phases) and stop-codon read through (‘RiboTools’). However, prior to any advanced Ribo-seq data processing, it is necessary to have standard pipelines for quality assessment of the experiments and specific ribosome footprint assignment to sequences.\n\nWe implemented the features for a standard Ribo-seq workflow in a pipeline entitled ‘RiboProfiling’. The pipeline takes an alignment file (BAM) as input, performs identification of the read offset, generates transcript and (multi-) codon coverage quantification data, and performs statistical analyses as well as graphical representations. Our pipeline is, to our knowledge, the most complete integration of a ribosome profiling standard analysis pipeline in an unique R framework. This includes the crucial step prior to quantifying ribosome footprints that consists in identifying the offset between Ribo-seq reads and the P-site of the ribosome. We have given special attention to this step as it is essential to correctly associate ribosome footprints with codon resolution. Depending on the RNA digestion protocol, the assignment of the ribosome must be made specifically either to the 5’, 3’ or the center of the read. To our knowledge, there is only one implementation for the determination of ribosome offset that was published so far. It was proposed in ‘riboSeqR’ as a metagene plot and the determination of the offset was only possible from the 5’ read end and for a particular read length. Our package offers several options, to compute the offset and recalibrate reads based either on the 5’ or the 3’ read ends. RiboProfiling also enables the graphical representation of ribosome density around the Translation Start Site (TSS) for multiple read lengths. This option allows to perform analysis for a single read length or on the merge of several read lengths. It enables to group all lengths sharing a same and unique offset value. The R/Bioconductor implementation provides an easy-to-use comprehensive set of functions that requires a minimal knowledge in R programming. The package contains a function entitled ‘riboSeqFromBAM’ that treats multiple Ribo-seq BAM files in parallel. The automated workflow generates report summary graphs and data quantification.\n\nWe illustrate the main features of the package using a Ribo-seq control sample from murine ES cells (GSM1655059), taken from a recently published ribosome profiling study using translation inhibitors (Popa et al., 2016). We then detail the analysis of ribosome accumulation on certain codons and tri-peptide motifs on a public dataset in Escherichia coli (GSE64488) (Woolstenhulme et al., 2015). The script for performing all these analyses is publicly available.\n\n\nMethods\n\nThe ‘RiboProfiling’ package, v.1.2.0 can be used with R 3.3.0 and Bioconductor version 3.3. The script needs a minimum memory limit in R of 3 Gb when analyzing tripeptide motifs. The package starts from alignment BAM files, from either Ribo-seq or RNA-seq experiments. We have validated BAM files from bowtie/tophat, Hisat2, STAR, and Lifescope (Solid), both single- or paired-end (for RNA-seq reads). Reads from rRNA, tRNA, and PCR duplicates (if unique molecular identifiers are available) should be removed from the BAM files before starting the analysis (see package vignette for details).\n\nAll analyses can be performed either through a call to a function called ‘riboSeqFromBAM’, or through a step by step approach. Figure 1 describes the workflow of the package starting from BAM files with reads mapped to the genome of interest. The first step in processing Ribo-seq reads is to select only those with match lengths compatible with standard ribosome footprints. The function ‘histMatchLength’ allows the visual inspection of read match sizes distribution across the BAM file (Figure 2.A), which should be enriched in reads of sizes between 20 to 40 nucleotides (Ingolia et al., 2009; Popa et al., 2016).\n\nA second particularity in the handling of Ribo-seq data comes from the shift existing between the extremities of the read (i.e. 5’ or 3’) and the P-site position of the ribosome. Our package allows the identification of an offset from the 5’ end of the read, but also from the 3’ end. The function ‘readStartCov’ computes the read frequency distribution centered on the translation start site (TSS) of the most expressed protein coding transcripts (by default the 3% most expressed). Based on this frequency distribution, the ‘plotSummarizedCov’ function enables the visual quantification of the offset between the reads and the ribosome P-site (Figure 2.B). In our Ribo-seq example, the 5’ read end is shifted 13 bp from the TSS. The innovation of this feature consists in the visualization of read lengths independently and as a summary figure.\n\nA, Histogram of read length distribution with ‘histMatchLength’ function. B, Frequency of reads around the TSS for reads sizes 26 to 28; this graph points to an offset of 13 bp between the read 5’ end and the ribosome P-site. C, Boxplots of Ribo-seq read coverage on the CDS, 5’ UTR and 3’ UTR regions of protein coding genes. D. PCA analysis of Ribo-seq coverage on codons.\n\nWhen computed, the offset can be applied on all reads based on the transcript referential with the function ‘countShiftReads’ and coverage on three different sequence features: 5’-UTR, coding sequences (CDS), and 3’-UTR (Figure 3.A). In Figure 2.C we observe that the majority of reads accumulate on the CDS of protein coding sequences and are practically lacking in the 3’ non-coding UTR regions. Ribosome footprints are also detectable in the 5’ UTR regions of protein coding genes, suggesting either the presence of coding upstream ORFs (Popa et al., 2016) or possible confounding information from missing annotations.\n\nA, Quantification of Ribo-seq read coverage on the CDS, 5’ UTR and 3’ UTR regions of protein coding genes. B. Quantification of Ribo-seq coverage on codons.\n\nFinally, our package provides quantification of ribosome footprints at codon resolution (Figure 3.B). A PCA analysis of codon occupancy can be performed and several graphical functions are implemented. In Figure 2.D we employed the ‘codonInfo’ and ‘codonPCA’ functions to analyze the codons accumulating ribosome footprints. As expected, the codon ATG is the most discriminant codon in the PCA analyses, since ribosome accumulation peaks are observed at the start codon of coding regions. Detailed descriptions with examples of the pipeline from BAM files to Ribo-seq reads quantification and processing are available in the vignette of our package: https://www.bioconductor.org/packages/release/bioc/vignettes/RiboProfiling/inst/doc/RiboProfiling.pdf.\n\n\nAnalysis of ribosome stalling on sequence motifs\n\n‘RiboProfiling’ can also be useful for analyzing ribosome occupancy on multi-codons motifs. Codons accumulating ribosome footprints are indicative of slowed ribosome progression (stalling) during the translation elongation process. The ‘RiboProfiling’ package offers several features for quantifying footprint accumulation on sequence motifs (ranging from one to three consecutive codons), performs principal component analyses, and allows graphical representation of those data.\n\nTo illustrate how ‘RiboProfiling’ can be used to explore the influence of sequence motifs (in this case tri-amino-acid sequences) on ribosome pausing, we analyzed an Escherichia coli Ribo-seq dataset (Woolstenhulme et al., 2015). We downloaded, filtered and mapped the reads of an efp-knockout sample (ΔEfp2, GSE64488), the elongation factor EFP being essential for the translation of polyproline motifs. Uniquely mapped reads from the resulting BAM file were analyzed with our ‘RiboProfiling’ package. After quality assessment of the reads size distribution, we quantified the offset between the 3’ end of the reads and the TSS for different alignment match sizes (Figure 4.A). We can clearly observe the 15 nucleotides offset that was reported by the authors for reads with alignment sizes >= 30 nucleotides. Smaller match lengths exhibited either a strong variation in the distribution of reads around the TSS (i.e. the 29 mers), or a different offset (i.e. offset of 20 for 21mers) (Figure 4.A). We selected the reads with alignment match sizes between 30 and 40 nucleotides and quantified codon coverage by positioning the ribosome P-site 15 nucleotides upstream of the read 3’ extremity.\n\nA, Sample ΔEfp2: percentage of read 3’ end coverage 20 nucleotides around the TSS. Three match sizes are represented: 21, 29 and 34 mers. B, Barplot of the average ribosome occupancy on PP(X) motifs in the ΔEfp2 sample, where X is any of the 20 possible amino acids.\n\nAn important stalling has been reported after the incorporation of two consecutive prolines (Pro-Pro) in the peptide chain. This stalling is highly dependent on the nature of the codon that follows the aminoacyl-tRNA reacting at the A-site (Woolstenhulme et al., 2015). Following the article’s analysis of stalling at PP(X) (Proline – Proline – 3rd codon) motifs, we used the ‘countShiftReads’ and ‘codonInfo’ functions to quantify the ribosome footprints on these motifs, in the ΔEfp2 sample. We then computed the pause score for all 20 possible PP(X) combinations in each ORF with more than 20 ribosome footprints, independently:\n\n\n\nwhere, ReadsPPX|ORF is the ribosome density of motif PPX in a given ORF; ReadsORF is the ribosome density on the ORF; NbrPPX|ORF is the number of time a given PPX motif is present in the ORF; LengthORF is the total length of the ORF. We averaged the ribosome occupancy for each PP(X) motif on all ORFs. Figure 4.B shows a strong stalling in sample ΔEfp2 when the ribosome encounters PPN, PPW, PPD, in agreement with their previous identification as pause sites in Escherichia coli (Woolstenhulme et al., 2015). A step by step R script implementing this entire analysis is provided at:\n\nhttp://genomique.info/data/public/RiboProfiling/scriptWoolstenhulme_Defp2.R.\n\n\nSummary\n\nOur ‘RiboProfiling’ Bioconductor package offers a collection of tools for Ribo-seq data analysis. It provides an unique, straightforward R implementation of a ribosome profiling pipeline from BAM, to P-site calibration, quantification of reads on sequence features, and codon coverage. The packages’ graphical features offer quality assessment and result representation across the analyses. Following the overview of Ribo-seq experiments with ’RiboProfiling, the output tables can then be easily integrated into more specialized dowstream analyses, either using more specialisez riboseq tools such as (XXX, YYY) or directly within R.\n\nWe highlighted here the features of our package in characterizing ribosome stalling at sequence motifs along ORFs based on an example dataset from Woolstenhulme et al. (Woolstenhulme et al., 2015). The workflow we propose for the analysis of ribosome occupancy on codon motifs using the ‘RiboProfiling’ package will most surely prove an useful asset in the context of recent ribosome profiling applications such as the detection of tumor sensitivity to differential amino acid depletion (Loayza-Puch et al., 2016).\n\n\nSoftware availability\n\nThe package is built in R (>=3.3.0) and freely available from Bioconductor website\n\n1. https://www.bioconductor.org/packages/release/bioc/html/RiboProfiling.html\n\n2. We provide an associated-script to the analyses in this paper at http://genomique.info/data/public/RiboProfiling/scriptWoolstenhulme_Defp2.R\n\nZenodo: scriptWoolstenhulme_Defp2.R, doi: 10.5281/zenodo.54567, (Popa A, 2016)\n\n3. GPL-3 licence",
"appendix": "Author contributions\n\n\n\nR.Waldmann, A.Popa, A.Paquet, and P. Barbry discussed the functions implemented in the package. A.Popa and K.Lebrigand developed the package. N.Nottet downloaded and performed the secondary analysis of Ribo-seq public datasets. A.Popa, A. Paquet, P. Barbry, R. Waldmann, K. Robbe-Sermesant wrote the article.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was developed by the Functional Genomics Platform at Nice Sophia Antipolis, a partner of the National Infrastructure France Génomique (ANR-10-INBS-09-03 and ANR-10-INBS-09-02) and PB’s group, thanks to supports by the Cancéropôle PACA and Commissariat aux Grands Investissements. RW was supported by Fondation ARC pour la recherche sur le cancer (SFI20121205973), and PB by ANR (ANR-12-BSVI-0023-02), Fondation pour la Recherche Médicale (DEQ20130326464) and labex Signalife (ANR-11-LABX-0028-01).\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\nBazzini AA, Johnstone TG, Christiano R, et al.: Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation. EMBO J. 2014; 33(9): 981–993. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFields AP, Rodriguez EH, Jovanovic M, et al.: A Regression-Based Analysis of Ribosome-Profiling Data Reveals a Conserved Complexity to Mammalian Translation. Mol Cell. 2015; 60(5): 816–827. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHardcastle TJ: riboSeqR: Analysis of sequencing data from ribosome profiling experiments. 2014. Reference Source\n\nIngolia NT, Ghaemmaghami S, Newman JR, et al.: Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. 2009; 324(5924): 218–223. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLegendre R, Baudin-Baillieu A, Hatin I, et al.: RiboTools: a Galaxy toolbox for qualitative ribosome profiling analysis. Bioinformatics. 2015; 31(15): 2586–2588. PubMed Abstract | Publisher Full Text\n\nLoayza-Puch F, Rooijers K, Buil LC, et al.: Tumour-specific proline vulnerability uncovered by differential ribosome codon reading. Nature. 2016; 530(7591): 490–494. PubMed Abstract | Publisher Full Text\n\nMichel AM, Mullan JP, Velayudhan V, et al.: RiboGalaxy: A browser based platform for the alignment, analysis and visualization of ribosome profiling data. RNA Biol. 2016; 13(3): 316–319. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO’Connor P, Andreev D, Baranov P: Surveying the relative impact of mRNA features on local ribosome profiling read density in 28 datasets. bioRxiv. 2015; 018762. Publisher Full Text\n\nPopa A: scriptWoolstenhulme_Defp2.R. Zenodo. 2016. Data Source\n\nPopa A, Lebrigand K, Barbry P, et al.: Pateamine A-sensitive ribosome profiling reveals the scope of translation in mouse embryonic stem cells. BMC Genomics. 2016; 17: 52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchafer S, Adami E, Heinig M, et al.: Translational regulation shapes the molecular landscape of complex disease phenotypes. Nat Commun. 2015; 6: 7200. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWoolstenhulme CJ, Guydosh NR, Green R, et al.: High-precision analysis of translational pausing by ribosome profiling in bacteria lacking EFP. Cell Rep. 2015; 11(1): 13–21. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "14285",
"date": "20 Jun 2016",
"name": "Olivier Namy",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 represents a useful package to analyze ribosome profiling data. We found that several important features of the Ribosome Profiling data processing are well implemented in this Bioconductor Package. Actually, the Kmer sizes distribution, the P-site location, the features (CDS, 5UTR, 3UTR) coverage distribution and the codon coverage are important and significant to explain Ribosome Profiling data and this package generates a great summary of these. However this package is not as complete as suggested by the authors. Indeed an important point is the periodicity of footprints that is not addressed here. Moreover it could be interesting to add the ability to detect which reading phase is indeed read. This should be feasible since the P-site is well defined.\n\nI include below a few comments for the authors.\nThe algorithm of the readStartCov function is not sufficiently described. The only one sentence found, does not give enough information to understand how it works. For instance, \"the read frequency distribution centered on the translation start site\": \"Centered\" needs more explanation. This is important, because using our own datasets we were not able to obtain any convincing results about the position of the P-site. The lack of information prevented us from understanding why.\n\nIn the Package Manual, when the aroundPromoter function is defined, the TSS is described as the \"Transcript Start Site\", which does not match with the \"Translational Start Site\" found in the article.\n\nThe pipeline requires an annotation file with a specific format (TxDb object). Users have to be aware of that if they want to use their own genome.",
"responses": []
},
{
"id": "14282",
"date": "21 Jun 2016",
"name": "Audrey M Michel",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRiboProfiling allows a number of ribo-seq specific analyses to be carried out using a single Bioconductor package in R and is supported by instructions in the corresponding reference and 'package' manuals. The automatic assignment of the P-site from either the 5' or 3' end of the ribosome footprint read is a useful feature. In addition the tool allows the quantification of the distribution of footprints across coding regions (CDS), 5' leader and 3' trailer regions at the metagene and individual gene transcript level. The authors use the reported stalling events in the Woolstenhulme et al., 2015 1 ribo-seq data to illustrate how RiboProfiling can be used to determine the ribosome occupancy on multiple codon motifs.\n\nOverall, the RiboProfiling package allows some ribo-seq specific analyses to be automatically carried out using the RiboProfiling 'quick command' or by using the step-by-step commands. However I would argue that RiboProfiling is not more complete than existing tools. There is no assessment of triplet periodicity for example. The quality assessment carried out by RiboProfilig is the distribution of footprint read lengths (Figure 1). While determining the read lengths in a ribo-seq dataset is a useful assessment, it is only one determinant of the quality of ribo-seq data. The RUST tool (O'Connor et al. 2015 2) as mentioned by the authors carries out normalisation of the data, but also allows for the quality of the ribo-seq and mRNA-seq data to be determined in terms of sequencing biases.\n\nNevertheless, RiboProfiling adds to the repertoire of publicly available ribo-seq tools which is useful to the community.\n\nQuestions:\n\nIn the manuscript the abbreviation TSS is used for Translation Start Site. However, on page 6 of the RiboProfiling manual “aroundPromoter: returns the genomic positions flanking the transcript start site (TSS) for the x% (3% default value) best expressed CDSs”. Should this be Translation start site?\n\nIt is not clear what the authors mean by tools such as (XXX, YYY) ' in the following sentence in the Summary section 'Following the overview of Ribo-seq experiments with ‘RiboProfiling, the output tables can then be easily integrated into more specialized dowstream analyses, either using more specialisez riboseq tools such as (XXX, YYY) or directly within R.' (There are also some typo errors in this sentence (dowstream; specialisez).\n\nWhile following the instructions in the RiboProfiling manual, there were several operations for which I was missing the required library (e.g. library(Rsamtools), library(GenomicAlignments), library(TxDb.Hsapiens.UCSC.hg19.knownGene), etc). While it might be obvious which additional libraries are required after running the command, it may help users if they were specified in the manual along with library(RiboProfiling).\n\nI ran the 'quick start' riboSeqFromBAM function on one of our own datasets and four plots were generated fine. However, the 'list of per ORF per codon coverage' was not output. Should this have been generated as stated in the manual?\n\nWhat read lengths are considered in the RiboProfiling 'quick command' approach for the offset determination? On our own dataset the sumUp offset plot was generated showing an offset of -13 (which corresponds to our own estimation). However, this plot did not show the individual read lengths. Are read lengths sharing the same offset value used?\n\nAll of the step-by-step commands in the user manual ran fine on our own dataset and the corresponding plots were generated. However, there was an error when I tried to run the accompanying scriptWoolstenhulme_Defp2.R to generate the barplot in Figure 4B of the average ribosome occupancy on PP(X) motifs in the E.coli dataset (see further). So unfortunately I was unable to verify this part of the manuscript.\n\nAccording to the manual, “consecutive motifs of 9 nucleotides (3 consecutive codons) overlap on 6 nucleotides. The Ribo-seq coverage is reported as the coverage on the 2nd codon in the motif considered as being in the P-site.” Is the average pause score in Figure 4B for ribosome occupancy of the second codon (P) or the last codon (X) or across the three codons?\n\nThe scriptWoolstenhulme_Defp2.R is hard-coded for the PPX motif and E.coli. I did not get to check if it can be easily adapted for other codon motifs in other datasets.\nBelow I provide a sub-set output of the scriptWoolstenhulme_Defp2.R with the error. If the sessionInfo() details can help troubleshoot the error, I can send it to the authors separately so as not clog up this report. …. Import genomic features from the file as a GRanges object ... trying URL 'http://genomique.info/data/public/RiboProfiling/Escherichia_coli_str_k_12_substr_mg1655_no_rRNA_no_tRNA.gtf'\n\nContent type 'unknown' length 2766800 bytes (2.6 MB) ================================================== downloaded 2.6 MB\n\nOK Prepare the 'metadata' data frame ... OK Make the TxDb object ... OK [knet_seek] SEEK_END is not supported for HTTP. Offset is unchanged. [bam_header_read] EOF marker is absent. The input is probably truncated. [knet_seek] SEEK_END is not supported for HTTP. Offset is unchanged. [bam_header_read] EOF marker is absent. The input is probably truncated.\nError in if (length(ixLengthPb)/length(testLength) >= 0.7) { :\n\nmissing value where TRUE/FALSE needed Calls: readStartCov -> readStartCov1Aln -> normRange Execution halted",
"responses": []
},
{
"id": "14283",
"date": "05 Jul 2016",
"name": "Ghislain Bidaut",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 started my review by trying to reproduce the vignette example. I also made sure that all documentation was present in the reference package PDF, which was the case.\nAs stated in the paper, the Riboprofiling package has to be installed under R version 3.3 minimum. I installed the latest version at the date of review, which was 3.3.1+ BioC 3.3.\nI started by the installation of the Riboprofiling package, which posed no problem.\nI am not proficient with existing pipelines, and did not verify if Riboprofiling is more complete than them.\nHowever, I found that an R implementation is nice since most users will be familiar with its installation, organisation and mode of use and won’t have to find other tools elsewhere.\nI have a few remarks on the installation and on the vignette example:\nI had to install the Bioconductor Rsamtools which was not installed alongside Riboprofiling, while other dependencies were installed.\nAfter running the covData <- riboSeqFromBAM(listInputBam, genomeName=\"hg19 »), I obtained the following warnings. Not sure if they impact the final result, but I nevertheless report them.\nWarning messages: 1: In riboSeqFromBAM(listInputBam, genomeName = \"hg19\") :\n\nparamScanBAM parameter is not a ScanBamParam object. Set to default NULL value!\n2: In doTryCatch(return(expr), name, parentenv, handler) :\n\n[knet_seek] SEEK_END is not supported for HTTP. Offset is unchanged. 3: In doTryCatch(return(expr), name, parentenv, handler) :\n\n[bam_header_read] EOF marker is absent. The input is probably truncated. 4: In countShiftReads(exonGRanges[names(cdsPosTransc)], cdsPosTransc, :\n\nParam motifSize should be an integer! Accepted values 3, 6 or 9. Default value is 3. 5: In doTryCatch(return(expr), name, parentenv, handler) :\n\n[knet_seek] SEEK_END is not supported for HTTP. Offset is unchanged. 6: In doTryCatch(return(expr), name, parentenv, handler) :\n\n[bam_header_read] EOF marker is absent. The input is probably truncated. 7: In countShiftReads(exonGRanges[names(cdsPosTransc)], cdsPosTransc, :\n\nParam motifSize should be an integer! Accepted values 3, 6 or 9. Default value is 3.\nIn the vignette page 5, I would add the statement\nlibrary(GenomicAlignments) before the readGAlignments call in order to make the code easier to work..\nVignete page 6. The instruction txdb <- TxDb.Hsapiens.UCSC.hg19.knownGene does not work. I do not have a TxDb.Hsapiens.UCSC.hg19.knownGene object in my session. I couldn’t go further in running the provided tutorial. Could you clarify on how to obtain that object ?\nApart from the vignette, the paper itself is well written and can be followed simply through. An analysis case of ribosome stalling study is presented through a complete example and R code (analysis of Woolstenhulme et al dataset). The code works without problem until the invocation of library(BSgenome.Ecoli.NCBI.K12.MG1655). I couldn’t get this package on the Bioconductor Web Site, and some clarification might be helpful for this step.\nMinor remark: The authors missed a couple of references in the last paragraph: (...) either using more specialisez riboseq tools such as (XXX, YYY) or directly within R.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1309
|
https://f1000research.com/articles/5-1305/v1
|
09 Jun 16
|
{
"type": "Review",
"title": "A step-by-step overview of the dynamic process of epitope selection by major histocompatibility complex class II for presentation to helper T cells",
"authors": [
"Scheherazade Sadegh-Nasseri"
],
"abstract": "T cell antigen receptors (TCRs) expressed on cytotoxic or helper T cells can only see their specific target antigen as short sequences of peptides bound to the groove of proteins of major histocompatibility complex (MHC) class I, and class II respectively. In addition to the many steps, several participating proteins, and multiple cellular compartments involved in the processing of antigens, the MHC structure, with its dynamic and flexible groove, has perfectly evolved as the underlying instrument for epitope selection. In this review, I have taken a step-by-step, and rather historical, view to describe antigen processing and determinant selection, as we understand it today, all based on decades of intense research by hundreds of laboratories.",
"keywords": [
"Epitope Mapping",
"Antigen Processing Machinery",
"immunodominance"
],
"content": "Introduction\n\nT cells and B cells are two major components of the adaptive and specific immune system. While B cells can recognize antigens as a whole via their B cell receptors, T cells can only see a processed form of antigens, that is, short peptide sequences bound to the proteins of major histocompatibility complex (MHC) class I and class II. There are also two major classes of T cells: cytotoxic T cells (Tc), which are restricted to MHC class I, express CD8 accessory molecules on their cell membranes, and function by killing their targets, and helper T cells, identified by restriction to MHC class II and expression of CD4 accessory molecules. Helper T cells function by producing cytokines that help B cells in antibody production and isotype switching, as well as helping CD8+ T cells to develop into memory cells. Helper T cells are divided into several subclasses, each having different functions1. CD8+ T cells are generally responsive to antigens such as viruses which have been endogenously expressed, while helper T cells present antigens taken up from exogenous sources. The machinery that best generates short peptides that bind to MHC molecules is present in antigen-presenting cells (APC). While a variety of cells might be able to process antigens under certain circumstances, dendritic cells (DC), B cells, and macrophages are considered professional APCs. Antigen processing for presentation by MHC class I follows a different biosynthetic pathway than that of MHC class II2,3. In the following sections, I focus on MHC class II, discussing different aspects of epitope generation and selection as assisted by the accessory molecules and processing enzymes that allow flawless completion of this complex process. At the end, I will briefly review attempts at identifying peptides that bind MHC molecules.\n\n\nMHC molecules have optimal structures for presenting antigens\n\nFor the presentation of antigen to helper T cells, APC must achieve an ambitious goal. One or few epitopes from a given antigen must be selected to fit stably and specifically in the groove of MHC class II. However, the number of possible epitopes to bind each MHC molecule is infinite, while each individual carries a maximum of six to eight MHC class II alleles. How is it possible for those few MHC molecules to bind peptides stably but non-specifically? The crystal structure of MHC class II, HLA-DR14, revealed two sets of interactions with the bound peptide: side chains of peptides interacting with five pockets (pockets 1, 4, 6, 7, and 9), and a series of 13 H-bonds that formed between peptide main chains and the non-polymorphic residues of the MHC groove. It appears that by adopting a combination of pockets that accommodate peptide side chains, the MHC molecule meets the specificity criterion, and by forming H-bonds, complex stability can be achieved.\n\nThe next challenging demand from the MHC class II structure is to ensure that peptides from the exogenous antigens bind to the groove of MHC II efficiently. The solution here is provided by evolving a peptide-binding groove that is highly flexible and susceptible to collapsing in the absence of a bound peptide5–7. I will write more about this topic later.\n\nThe flexibility of the groove is a theme that I shall revisit throughout this review. To appreciate this concept, the readers of this review are likely to benefit from a brief history of peptide binding to MHC class II as part of its folding. Harden McConnell’s group was the first to realize that there were kinetic and structural intermediates in peptide binding to MHC II8–11. Using a simple SDS-PAGE assay where samples were kept at room temperature, the team demonstrated that naturally formed peptide/MHC (pMHC) complexes, purified from APC, migrated differently if peptides were dissociated. A loosely bound pMHC, or a peptide free MHC molecule, migrated as a slower migrating species that was named floppy dimers, relative to a faster migrating species called compact dimers. Compact dimers were shown to contain peptide, and unstably bound pMHC dissociated into single chains in SDS-PAGE (SDS sensitive)10,11. Importantly, when peptides that could form stable complexes with MHC II molecules were added back to MHC II, the partially unfolded floppy dimers and the dissociated chains reverted to compact conformations6,7. It was of great significance that the in vitro findings were confirmed in cells. In pulse-chase experiments, analyzed by SDS-PAGE, newly synthesized MHC II molecules that were not in complex with peptides from exogenous sources (pulse) dissociated into single chains, whereas MHC class II molecules that had formed complexes with exogenous peptides (chase) migrated as SDS-stable dimers12,13. By this criterion, class II molecules were shown to associate with peptides in the endocytic route prior to cell surface expression, a process that requires proteolytic digestion of the protein antigens13. It was also shown that SDS stability did not always correlate with the stability of pMHC complexes; altered MHC mutants bound peptides loosely yet formed the characteristic SDS-stable conformation14. Those original observations have been confirmed through numerous techniques over two decades of research by independent laboratories15–19.\n\nThe remarkable characteristic of MHC class II to resist SDS denaturation when in complex antigenic peptides allowed new discoveries that revealed steps in MHC class II synthesis, association with invariant chain (Ii), exposure to antigen-processing enzymes, MHC II trafficking, interaction with accessory molecules, peptide binding and editing, and more, as discussed below.\n\n\nAntigen-processing machinery\n\nAntigen presentation to CD4+ T cells begins by the uptake of exogenous antigens by APC and their processing by proteolytic enzymes, mainly different cathepsins (Cat). The process involves transfer through a series of vesicular subcompartments containing suitable denaturing environments, a variety of accessory molecules and molecular chaperones, as well as cathepsins20. Cathepsins present in processing compartments contribute by cutting and trimming of the protein antigens.\n\nAntigen-processing proteases, or cathepsins, are amongst the most significant contributors to antigen processing and act as exoproteases, or endopeptidases21. Expression levels and the activity of cathepsins are highly regulated in different cell types and activation states. Historically, two main roles have been described for cathepsins in antigen processing: to cleave off Ii and to process protein antigens. A new important function for cathepsins in the selection of immunodominant epitopes has recently been described and will be discussed later22. Some of the most extensively studied cathepsins are CatB, CatD, CatL, and CatS23–27. CatS was reported to be involved in Ii cleavage and antigen processing28–31. Recent studies by Kim et al.22 using a cell free processing system showed that inclusion of only three cathepsins (CatB, CatH, and CatS) was sufficient to mimic the processing conditions necessary to produce the immunodominant epitopes from several protein antigens. It is of note that cathepsins involved in antigen processing require acidic pH for their proteolytic function, which itself is highly regulated. Indeed, DC maturation promotes activation of vacuolar proton pumps and enhances lysosomal acidification32.\n\nUpon synthesis, every allele of the MHC II heterodimers forms complexes with a third nonpolymorphic chain, called class II Ii, which acts as a chaperone in folding among its several other functions. The Ii was first discovered by Jones and McDevitt, and was found to bind to all MHC II alleles33. After intense research by numerous laboratories, it became clear that Ii acts as a chaperone for the newly synthesized MHC II34. Its structure is rather segmented, each having a different function. Using nuclear magnetic resonance (NMR) techniques, Jasanoff et al.35–37 reported that a soluble recombinant Ii in complex with MHC II was mainly disordered except for two regions, one that included a region of 24 amino acids corresponding to the class II-associated Ii peptide (CLIP) and the other which participated in trimerization of the Ii to form nonomeric assemblies. The CLIP region binds in the peptide-binding groove of class II molecules in the endoplasmic reticulum (ER) and remains bound in cleaved form in the peptide-loading compartment, where the rest of the Ii is cleaved off by cathepsins38–41. Another important function of Ii is to target the newly synthesized MHC class II to the proper endocytic compartments42, where it intercepts with protein antigens. The specialized endosomal compartments, called MIIC or CIIV43–47, were discovered as lysosome-like compartments which contained all necessary machinery for the processing of antigen and the optimal binding and selection of the peptides for presentation to the T cells48. These vesicles are dense membranous structures that fall between the early endosomes and lysosomes in density, as well as their denaturing environment to include acidic pH49, denaturing and proteolytic enzymes.\n\nIn addition to its other chaperoning functions, binding of Ii to MHC II was originally considered a means of preventing unproductive binding of peptides present in the ER39. However, when the first Ii knockout mice were reported, it turned out that the MHC II molecules of Ii-deficient mice did not bind as many peptides as did the MHC II molecules of Ii-sufficient mice50,51. With better understanding of the flexibility and instability of the peptide-binding groove in the absence of a bound peptide52–56, it became clear that an unappreciated function of the CLIP region is that it acts as a surrogate peptide for shaping the MHC II groove. These studies demonstrated that the MHC class II groove collapses in the absence of a bound peptide and that a poor binding peptide, such as CLIP, maintains the groove in proper conformation. Upon dissociation of CLIP, a peptide-receptive conformation is generated that can scan peptides or unfolded proteins in the antigen-processing compartments55,56. Ii shuttles the MHC II molecules to MIIC, where Ii is proteolyzed by different cathepsins, including CatS, until only the CLIP fragment remains bound in the MHC II peptide-binding groove26,31,57,58. CLIP must then be exchanged for exogenous peptide, a function best performed by the accessory molecule HLA-DM in humans or H2-M in mice (DM, from now on)59.\n\nDM is a non-polymorphic MHC II-like molecule that does not bind peptides itself60 but is necessary for the efficient displacement of CLIP from the MHC groove59,61–69. The significance of DM in antigen presentation was first discovered through the observation that some APC lines did not process protein antigens for presentation to specific T cells. Those cell lines were found to have defective DM genes70. Later, it was discovered that HLA-DR molecules from an antigen-processing mutant cell line were occupied with invariant chain peptides61,71. Hence DM was critical for the removal of CLIP and its exchange for the exogenous peptides.\n\nMechanism of DM function. Understanding the mechanism of the function of DM posed a problem for a long period of time, as it was generally believed that DM dissociated all bound peptides from MHC II molecules. This concept created a dilemma: how could any peptide remain bound in the groove of MHC II when every peptide was susceptible to dissociation? The problem was partially solved by the finding that not all peptides were equally susceptible to DM-mediated dissociation72,73. Certain peptides that would fit the MHC II groove and formed a rather rigid or compact conformation remained resistant to DM-mediated dissociation74–84. It was proposed that DM functions by recognizing conformations of pMHC II complexes that vary based on the nature of the bound peptides. For the best-studied MHC II molecule, HLA-DR1, it is well established that P1 interaction is the key determinant of pMHC II complex stability14,52,85 and that peptides interacting non-optimally in the P1 pocket are highly susceptible to DM-mediated peptide exchange15,74,75,80,82,86,87. DM interaction induces major conformational alterations in the P1 area of the MHC II groove, leading to destabilization of the bound peptide and preventing the formation of H-bonds, hence peptide dissociation. When peptide is released, a peptide-receptive MHC II is generated74,86, which can quickly sample a large pool of sequences from the available proteins.\n\nThe significance of P1 in interaction with DR1 was demonstrated by a mutagenized DR1 that expressed a partially filled P1 pocket and failed to interact with DM15,74,82. The mutant molecule, DR1(bG81Y), carrying a single amino acid change from G to Y, was constitutively peptide receptive and migrated as compact dimers in gentle SDS-PAGE (Figure 1). Indeed, the DR1(bG81Y) molecule resembled murine I-Ek, which has a shallow pocket 115 and itself is resistant to the DM editing function. In agreement with the structural characteristics of I-Ek, DM knockout haplotype K mice did not show the characteristic defects in peptide binding and occupancy with CLIP associated with H-2b mice88. Thus, DM can only affect peptide exchange in MHC II alleles of certain structural requirements89.\n\nSubstitution of glycine for tyrosine at position 86 of DR1 beta chain generates a P1 pocket that is partially filled and resistant to DM-induced effects14.\n\nWith the solution of the crystal structure of the DM/DR complex using a cleverly designed DR1/peptide complex that allowed for the DR1 groove to remain open, it was established that DM would bind the P1 pocket of HLA-DR molecules if empty and would remain bound until a P1 filling peptide bound the groove17,84,90. The above findings were complemented by the measured thermodynamics of peptide binding to DR1, indicating that a greater entropic penalty, versus a smaller penalty, was associated with structural rigidity rather than with the flexibility of the pMHC complexes87. Consistent with the previous reports, the authors found that DM senses flexible complexes, in which the P1 area residues are rearranged at a higher frequency than in more rigid complexes. Moreover, a new and unexpected observation reported that conformational changes in the P1 area could be negated if the P9 pocket anchor residue of peptide was substituted for a stronger binding residue16. The findings suggest that an overall dynamic MHC II conformation, in addition to P1 pocket occupancy, determines susceptibility to HLA-DM-mediated peptide exchange and provides a molecular mechanism for HLA-DM to efficiently target poorly fitting pMHC II complexes, editing them for more stable ones. Hence, in addition to the removal of CLIP, DM helps in shaping epitope selection (more details to follow).\n\nBiological significance of DM. As discussed earlier, DM plays an important role in selecting the right peptides that can stay in the groove of MHC II long enough for T cell recognition91. This characteristic of DM contributes to T cell immunity in a significant way. Lymphocytes usually respond to a small proportion of the potential determinants on a protein antigen defined as “immunodominant”92. Immunodominant epitopes are the essential targets of the immune response against infectious diseases, cancer, autoimmune diseases, and allergy. Consequently, much attention has been devoted to the understanding of epitope selection and immunodominance. However, in spite of the complexities of antigen processing and presentation, T cell epitope discovery has been a challenging task. Some of the factors contributing to immunodominance are described below.\n\nEpitope accessibility and its relation to immunodominance. Among many contributing factors to an epitope gaining immunodominance is how accessible the location of sequence is to the groove of the MHC II molecule and/or to the processing enzymes93. The denaturing environment in the antigen processing compartments (acidic pH and reducing conditions) helps to partially unfold protein antigens to reveal hidden epitopes. Of particular interest is a specialized enzyme, gamma-interferon-inducible lysosomal thiol reductase (GILT), that releases disulfide bonds in proteins94, making denaturation more efficient. In support of the significance of GILT in the release of dominant epitopes is the fact that GILT-deficient mice failed to present buried determinants of hen egg lysozyme (HEL) and an HA protein of influenza; HEL and HA both have four disulfide bonds95,96. Support of the “epitope accessibility” model for immunodominance comes from accumulated evidence that many of the naturally selected epitopes localize on flexible strands of protein antigens93 or at the C- or N-terminus of protein antigens97–99. For a more comprehensive review on the subject of accessibility, the readers are referred to 102.\n\nOne question that might come to the mind of readers is how is it that the MHC II and their accessory molecules are not denatured in such an aggressive environment? It is of note that acidification of the antigen processing compartments in DC is developmentally regulated. Hence, the vacuolar proton pump that acidifies MIIC and activates cathepsins for processing of internalized antigens is activated only upon DC maturation32. Also of importance is that MHC II and DM molecules resist denaturation and cleavage100 by the harsh acidic pH and proteolytic conditions likely present in the late endosomes.\n\nA cell free reductionist antigen processing system. A need for epitope accessibility together with the open-ended groove of MHC class II hint at binding of MHC II to the whole antigen rather than precut peptides. While there have been several examples of MHC II binding to full length antigens101–103, the prevailing dogma assumes that peptides are cut first, and then binding to MHC II and selection by DM takes place104. However, direct evidence in support of binding of full length protein to MHC II and determinant selection by DM was put forth by the design and use of a reductionist cell free antigen processing system, which documented that full-length proteins, or a mixture of protein fragments, could be processed and the immunodominant epitopes could be selected by a minimal number of ingredients100 (Figure 2). The components of this minimalist system include MHC class II, HLA-DR, full-length denatured protein antigen, three processing enzymes, cathepsins S (an endopeptidase), B, and H (exopeptidases), and HLA-DM, all placed in a tube in acidic pH. After allowing time for processing of the antigen, peptide binding, and DM editing, DR molecules, now bound to the selected epitopes, are immunoprecipitated and the bound peptides are released by exposure to low pH and are then subjected to mass spectrometry. In the following steps, the identified peptides, which usually are not very many, will be tested for immunogenicity in HLA-DR1-expressing Tg mice immunized with the full-length protein antigens. The results were quite pleasing: peptides identified by the reductionist system were immunodominant epitopes because they recalled nearly full T cell responses. Importantly, even when tested in human volunteers, the peptides identified by the reductionist system proved to accurately reflect antigen processing in human APCs100. It is significant that the immunodominant epitopes were identified when DM was included in the system whereas, in the absence of DM, other non-dominant epitopes were also found among the eluted peptides.\n\nPurified MHC class II and accessory molecules are exposed to full-length antigens and cathepsins under denaturing conditions. MHC class II molecules are then isolated and subjected to peptide elution and mass spectrometry100.\n\nThe results from the reductionist system suggested that DM plays a key role in the selection of the immunodominant epitopes from exogenous antigens22,100,105,106. In a later extensive study, Yin et al. compared affinity, intrinsic dissociation half-life, and DM-mediated dissociation half-life as well as two epitope prediction algorithms (more below) for many peptides derived from the entire Vaccinia genome for inducing CD4+ T cell responses. The results confirmed that pMHC II complex kinetic stability in the presence of DM was the determining factor for distinguishing the immunodominant epitopes from the non-dominant bound peptides107. In agreement with results from the reductionist system, these analyses demonstrated that DM editing governs peptide immunogenicity by favoring the presentation of peptides with greater kinetic stability. However, it is of note that not all stable pMHC complexes are immunodominant. Moreover, autoimmune epitopes may or may not be resistant to DM-mediated dissociation22.\n\nThe use of the cell free reductionist system also enabled the authors to gain a new understanding of dominant epitope selection22,105,106,108. The authors showed that peptides derived from pathogens, or autoantigens, behaved differently in response to DM. For autoantigens, resistance to DM-mediated dissociation was not a required criterion, whereas for pathogen-derived dominant epitopes, DM resistance was a crucial factor. Immunodominance emerged as a result of the combined effects of DM and the antigen processing cathepsins. Autoantigen-derived immunodominant epitopes were resistant to digestion by cathepsins in the system, whereas pathogen-derived epitopes were sensitive. As such, sensitivity to cathepsins necessitated the capture of pathogen-derived epitopes by MHC II prior to cathepsins processing, and resistance to DM-mediated-dissociation preserved those epitopes from pMHC release and degradation22. The overall findings demonstrated that immunodominance is established by the higher relative abundance of the selected epitopes that survive cathepsins digestion either by binding to MHC II and resisting DM-mediated-dissociation or by being chemically resistant to cathepsin degradation. Non-dominant epitopes were found to be sensitive to both DM and cathepsins22 (Figure 3). Consistent with the autoimmune epitopes being resistant to proteolysis is the finding that large numbers of peptides derived from autoantigens have been identified in normal pre-nodal afferent human lymph109,110. The lymph peptidome must have resisted the variety of catabolic enzymes present in tissues, the function of which remains to be understood.\n\nInfluenza-derived HA protein (left) is captured as full-length denatured protein or large fragments by MHC class II, edited by DM, and then is exposed to cathepsin digestion. An example of an auto-antigen, collagen (right), is first cut into short peptides and then binds MHC class II; it may be either sensitive or resistant to DM-mediated editing22.\n\nPossible role of DM in the quality of the peptide/MHC II complex. Intriguingly, there are reports documenting that some autoimmune T cells might discriminate among peptides that form complexes with MHC II in the presence or absence of DM. A clear example has been pioneered by Unanue and colleagues, who showed two types of T cells: type A that recognize pMHC generated by intracellular processing machinery including DM and type B T cells that recognize pMHC formed in the absence of DM111,112. Of outstanding interest is that autoreactive CD4+ T cells specific for an insulin peptide were type B T cells; they did not recognize the insulin protein when processed by APC and, as such, could not have been deleted during thymic education113. These findings suggest that the topology of the complexes formed in the presence or absence of DM might be different. The findings of the Unanue and Sadegh-Nasseri laboratories hint at the possibility of a different path for antigen processing for autoimmune epitopes. As discussed, autoimmune epitopes may or may not be sensitive to DM-mediated dissociation, and they are highly resistant to the proteases in antigen processing22. Hence, it is very likely that for some autoimmune diseases, autoantigens are generated in an extracellular matrix where many proteases are already at work. The core epitopes that survive such a protease-rich milieu may get a chance to bind to the empty MHC II molecules expressed on APC membranes or in the early endosomal compartment where DM is not active114. Such complexes would not be edited by DM and would fit the required ligand characteristic for type B T cells. Alternatively, some of the larger antigen fragments might be processed in the early endosomes where DM does not contribute to peptide editing, leading to the generation of type B pMHC complexes.\n\nIn addition to DM, another non-classical MHC class II accessory molecule, HLA-DO, H2-O in mice, DO from now on, is known to play a role in peptide exchange115–117. Of importance, DO has restricted tissue expression; it is mainly expressed in B cells and thymic medullary epithelium, where thymic deletion takes place. In addition, certain subsets of DCs express DO under different conditions. Cellular trafficking of DO depends on DM. Understanding how DO contributes to antigen processing has been a challenge for decades. Two recently solved crystal structures, DM/DO and DM/DR1, suggested that the DM/DO interface is shared with the DM interface with DR190,118. These findings were interpreted to imply that DO might act as a competitive inhibitor of DM in interaction with DR. While this model has previously been advocated117, peptide binding association and dissociation kinetics conducted in the presence of DM, and/or DM/DO, put forward an alternative mechanism115,116. It was shown that DO binds to DR molecules. Rather than inhibiting DM, it was demonstrated that DO works together with DM to increase the binding of peptides that formed DM-resistant complexes with DR, while reducing the binding of peptides that are DM sensitive. Furthermore, the positive and negative effects of DO on peptide binding were shown to be restricted to the association phase, as the peptide dissociation phase remained unaffected by DO. Interestingly, DO could only bind to a peptide-receptive rather than peptide-occupied DR1. Because DO is always in complex with DM, and DM works by generating a peptide-receptive conformation, the authors proposed a model to suggest that DM might dissociate pMHC, leading to a peptide-receptive DR that can be stabilized by DO. Thus, DO and DM work in synergy for optimizing peptide exchange and for selecting the DM-resistant peptides. The combined efforts of all the molecules discussed above, and perhaps others whose identities are yet to be discovered, lead to an impeccable selection process for the immunodominant epitopes for MHC II groove occupancy and transport to the APC external membrane for CD4+ T cell stimulation.\n\n\nSearch for the immunodominant epitopes\n\nClearly, finding peptide epitopes that bind to MHC molecules and represent a given antigen is highly desirable for use in therapeutics and vaccine designs. A variety of methods have been used for this purpose. Among those is the screening of hundreds of overlapping synthetic peptides that span the entire sequence of an antigen of interest for binding to MHC II molecules. The screening may involve biophysical methods to measure MHC II binding, T cell readout assays, or tetramer-guided epitope mapping. Hundreds of tetramers would be made using hundreds of overlapping peptides for detection of the T cells reactive to the antigen119. These methods are generally labor intensive, costly, and often insufficient because, with the exception of tetramer-guided mapping, they do not take into account steps involved in the processing of antigens as it happens in the cellular environment. In the identification of autoimmune epitopes, the task is even more arduous because autoimmune epitopes, in addition to the characteristics discussed above, often include post-translational modifications119, which makes screening of the peptides based on the amino acid sequences of the proteins rather hopeless.\n\nComputational approaches are popular alternative methods for predicting possible epitopes that bind MHC class II molecules with high affinity. The guiding principle in all computational methods is the structural information available on different MHC grooves. Although somewhat successful for predicting MHC class I epitopes, computer prediction algorithms have been generally unsuccessful in identifying CD4+ T cell epitopes120–122. The open-ended MHC class II groove versus the better-defined pocket-fitting residues for MHC class I adds to the complexity of structure-based predictions exponentially. In addition, there is no computational way yet available for predicting how DM and DO would contribute to the epitope selection107.\n\nThe next popular method in epitope discovery is the use of mass spectrometry. Mass spectrometry for the identification of peptides eluted from MHC class I and class II was first reported in the early 1990s123 and required large amounts of purified MHC molecules. Thousands of self-peptides are generally eluted from MHC molecules isolated from even uninfected APCs. In recent years, because of the great progress in the development of highly sensitive instruments for mass spectrometry, the need for high quantities of starting numbers of APCs (~1011) has been significantly reduced124. However, for an accurate determination of the dominant epitopes by peptide elution, it is necessary to utilize quantitative mass spectrometry because immunodominant epitopes are often displayed at the highest prevalence22, yet quantitative mass spectrometry has its own associated extreme complexities125.\n\n\nConcluding remarks\n\nAs discussed above, antigen processing is a complex multistep process that has evolved for the identification of the best-fitting epitopes for T cell recognition and functions. A number of chaperones together with the uniquely evolved MHC class II molecular structure, which requires a peptide as part of its fully folded state, contribute to this ultimate goal. While much has been learned over the past decades about antigen processing and presentation, because of the complexities involved, a successful peptide prediction strategy has yet to be discovered. The divergent paths for the processing of proteins of exogenous versus self-antigens open up new fields to explore. Understanding the biology of DO and its effects on the development of autoimmune diseases is another area that has remained challenging. Finally, the expression of MHC II upon T cell activation and its impact on immune responses begs further research. We can only hope that current and future research will focus on these unanswered questions.",
"appendix": "Competing interests\n\n\n\nThe author declares that she has no competing interests.\n\n\nGrant information\n\nSupported by grants R01AI063764 and R21 AI101987 from NIAID, NIH.\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\nI wish to dedicate this review to the memories of my mentors, Eli Sercarz and Harden McConnell, who were instrumental in conceiving and promoting many of the concepts discussed here. In addition, I sincerely thank all of the members of my laboratory whose hard work has led to some of the insights described in this review. Finally, I extend my gratitude to the reviewers of the manuscript who made many good suggestions that led to refining this review.\n\n\nReferences\n\nZhu J, Paul WE: Heterogeneity and plasticity of T helper cells. Cell Res. 2010; 20(1): 4–12. 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PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSadegh-Nasseri S, Kim A: MHC Class II Auto-Antigen Presentation is Unconventional. Front Immunol. 2015; 6: 372. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClement CC, Cannizzo ES, Nastke MD, et al.: An expanded self-antigen peptidome is carried by the human lymph as compared to the plasma. PLoS One. 2010; 5(3): e9863. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClement CC, Rotzschke O, Santambrogio L: The lymph as a pool of self-antigens. Trends Immunol. 2011; 32(1): 6–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLovitch SB, Walters JJ, Gross ML, et al.: APCs present A betak-derived peptides that are autoantigenic to type B T cells. J Immunol. 2003; 170(8): 4155–60. PubMed Abstract | Publisher Full Text\n\nLovitch SB, Esparza TJ, Schweitzer G, et al.: Activation of type B T cells after protein immunization reveals novel pathways of in vivo presentation of peptides. J Immunol. 2007; 178(1): 122–33. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMohan JF, Levisetti MG, Calderon B, et al.: Unique autoreactive T cells recognize insulin peptides generated within the islets of Langerhans in autoimmune diabetes. Nat Immunol. 2010; 11(4): 350–4. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSantambrogio L, Sato AK, Carven GJ, et al.: Extracellular antigen processing and presentation by immature dendritic cells. Proc Natl Acad Sci U S A. 1999; 96(26): 15056–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPoluektov YO, Kim A, Sadegh-Nasseri S: HLA-DO and Its Role in MHC Class II Antigen Presentation. Front Immunol. 2013; 4: 260. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPoluektov YO, Kim A, Hartman IZ, et al.: HLA-DO as the optimizer of epitope selection for MHC class II antigen presentation. PLoS One. 2013; 8(8): e71228. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDenzin LK, Cresswell P: Sibling rivalry: competition between MHC class II family members inhibits immunity. Nat Struct Mol Biol. 2013; 20(1): 7–10. PubMed Abstract | Publisher Full Text\n\nGuce AI, Mortimer SE, Yoon T, et al.: HLA-DO acts as a substrate mimic to inhibit HLA-DM by a competitive mechanism. Nat Struct Mol Biol. 2013; 20(1): 90–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDarrah E, Rosen A, Giles JT, et al.: Peptidylarginine deiminase 2, 3 and 4 have distinct specificities against cellular substrates: novel insights into autoantigen selection in rheumatoid arthritis. Ann Rheum Dis. 2012; 71(1): 92–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDoolan DL, Southwood S, Freilich DA, et al.: Identification of Plasmodium falciparum antigens by antigenic analysis of genomic and proteomic data. Proc Natl Acad Sci U S A. 2003; 100(17): 9952–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRammensee H, Bachmann J, Emmerich NP, et al.: SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999; 50(3–4): 213–9. PubMed Abstract | Publisher Full Text\n\nPeters HO, Mendoza MG, Capina RE, et al.: An integrative bioinformatic approach for studying escape mutations in human immunodeficiency virus type 1 gag in the Pumwani Sex Worker Cohort. J Virol. 2008; 82(4): 1980–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHenderson RA, Michel H, Sakaguchi K, et al.: HLA-A2.1-associated peptides from a mutant cell line: a second pathway of antigen presentation. Science. 1992; 255(5049): 1264–6. PubMed Abstract | Publisher Full Text\n\nClement CC, Becerra A, Yin L, et al.: The Dendritic Cell Major Histocompatibility Complex II (MHC II) Peptidome Derives from a Variety of Processing Pathways and Includes Peptides with a Broad Spectrum of HLA-DM Sensitivity. J Biol Chem. 2016; 291(11): 5576–95. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nOng SE: Whole proteomes as internal standards in quantitative proteomics. Genome Med. 2010; 2(7): 49. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "13410",
"date": "09 Jun 2016",
"name": "Malini Raghavan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13407",
"date": "09 Jun 2016",
"name": "Nilabh Shastri",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13409",
"date": "09 Jun 2016",
"name": "Bénédicte Manoury",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13406",
"date": "09 Jun 2016",
"name": "Laura Santambrogio",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1305
|
https://f1000research.com/articles/5-1268/v1
|
08 Jun 16
|
{
"type": "Research Article",
"title": "DNA barcoding of Clarias gariepinus, Coptodon zillii and Sarotherodon melanotheron from Southwestern Nigeria",
"authors": [
"Mofolusho O. Falade",
"Anthony J. Opene",
"Benson Otarigho",
"Anthony J. Opene",
"Benson Otarigho"
],
"abstract": "DNA barcoding has been adopted as a gold standard rapid, precise and unifying identification system for animal species and provides a database of genetic sequences that can be used as a tool for universal species identification. In this study, we employed mitochondrial genes 16S rRNA (16S) and cytochrome oxidase subunit I (COI) for the identification of some Nigerian freshwater catfish and Tilapia species. Approximately 655 bp were amplified from the 5′ region of the mitochondrial cytochrome C oxidase subunit I (COI) gene whereas 570 bp were amplified for the 16S rRNA gene. Nucleotide divergences among sequences were estimated based on Kimura 2-parameter distances and the genetic relationships were assessed by constructing phylogenetic trees using the neighbour-joining (NJ) and maximum likelihood (ML) methods. Analyses of consensus barcode sequences for each species, and alignment of individual sequences from within a given species revealed highly consistent barcodes (99% similarity on average), which could be compared with deposited sequences in public databases. The nucleotide distance between species belonging to different genera based on COI ranged from 0.17% between Sarotherodon melanotheron and Coptodon zillii to 0.49% between Clarias gariepinus and C. zillii, indicating that S. melanotheron and C. zillii are closely related. Based on the data obtained, the utility of COI gene was confirmed in accurate identification of three fish species from Southwest Nigeria.",
"keywords": [
"DNA Barcode",
"Clarias",
"Tilapia",
"COI gene",
"16S rRNA"
],
"content": "Introduction\n\nThe use of a globally recognized short DNA sequence, DNA barcode, for identification of species has gained global support as an applicable tool for species identification, particularly with respect to fishes as coordinated by the fish barcode of life (FISH-BOL; www.fishbol.org)1. Fish biodiversity in tropical Africa demonstrate an amazing variety of shape, size, and color. However, many of these fishes are under immense pressure from overfishing and climate change. In addition, the lack of appropriate methods of identification has limited our ability for classification, thus limiting the information available for fishery management2. About 65% of fishes captured worldwide have been identified to species level ranging from about 90% in temperate areas to less than 40% in tropical regions3. However, there is the need to identify more fish species from Africa, where there is a dearth of information on indigenous fish species4. The paucity of taxonomic data on local fish can be ascribed to the limitations imposed by traditional-based morphological identification, which can be confusing and unreliable due to problems of intraspecific, phenotypic and cryptic variation often overlapping among sister taxa in nature5. Consequently, the limitations imposed by morphological identification, have made the use of molecular diagnostic tools as a prerequisite for effective species identification6. DNA-based identification techniques have been developed and shown to be analytically important for characterization of organisms. DNA taxonomic techniques such as DNA barcoding have been useful for species identification and description7. DNA barcoding has been used to identify species and is important in characterizing biological diversity. This technique involves the amplification and sequencing of short universal molecular tags from a highly conserved gene. The mitochondrial cytochrome oxidase I (COI) gene is commonly used for this purpose8. The method is rapid, accurate and useful in delineating differences between species9. Consequently, the mitochondrial genes COI and 16S have been successfully employed in species identification based on DNA barcodes10 and a series of barcoding projects involving various organisms from different geographic regions is available at the public barcode library (www.barcodinglife.com)11.\n\nDespite the large information that exists for temperate fishes12, there are rather limited data for tropical fishes especially from Nigeria. A report by Nwani and colleagues employed DNA barcoding to discriminate freshwater fishes from Southeastern Nigeria where they provided a river system-level phylogeographic resolution of some of the fishes identified in their study13. Recently, Nwakanma et al.,14 also employed DNA barcoding in studying genetic diversity of fishes from Ugwu-omu Nike river of Enugu State, also in Southeastern Nigeria. It is therefore imperative to apply these tools to fishes from other areas in the country. Two of the most common freshwater fishes consumed by the population are from the genus Clarias and Tilapia (Coptodon)15,16. Of these, the most utilized in aquaculture and fish farming is C. gariepinus. Tilapia, belonging to the family Cichlidae, is a highly diverse group of more than 70 species found in Nigeria15. However, interbreeding of these fishes makes species delineation through morphology difficult.\n\nConsequently, in this study, COI and 16S genes were employed in performing an identification and diversity study of C. gariepinus, Coptodon zillii and Sarotherodon melanotheron from Southwestern Nigerian freshwater bodies.\n\n\nMaterials and methods\n\nEthical approval for animal experiments is given based on institutional guidelines. Collection of fish specimens and all laboratory experiments were thus performed in strict accordance with the recommendations of the University of Ibadan Ethical Committee on the use of laboratory animals for research.\n\nThree fish species, C. gariepinus, C. zillii and S. melanotheron were obtained with the aid of a local fisherman from Odooba River and Asejire Lake in Southwestern Nigeria. Both sites are tropical and characterized by two annual seasons of wet (April–September) and dry (October to March) seasons. The former site lies between 3.9°E and 7.4°N close to the University of Ibadan, Oyo State. Dead fish samples were collected and transported on ice to the Hydrobiology and Fisheries Laboratory of the Department of Zoology, University of Ibadan, where all fish specimens were morphologically identified to the species level by fish taxonomists using identification keys described by Olaosebikan and Raji15. Thereafter, fish specimens were preserved at –80°C until DNA extraction.\n\nExcised muscle tissue samples from the side of each fish were used to extract DNA. DNA was isolated using the QIAamp® DNA mini kit (QIAGEN, USA), following the manufacturer’s instructions. The concentrations and purity of the extracted DNA were estimated using a Nanodrop spectrophotometer (Nanodrop® ND -1000- NanoDrop Technologies, Inc.). Extracted DNA was visualized on a 2% agarose gel stained with ethidium bromide.\n\nTo amplify from the 5/ region approximately 570 bp fragment of the 16S rRNA gene and 655 bp of the COI gene, PCR reactions were conducted using the following primers: for 16 rRNA, 16SarL-F (5′-CGC CTG TTT ATC AAA AAC AT-3′) and 16SbrH-R (5′-CCG GTC TGA ACT CAG ATC ACG T-3′); for COI, FishF1 (5/-TCA ACC AAC CAC AAA GAC ATT GG CAC-3/) and FishR1 (5/-TAG ACT TCT GGG TGG CCA AAG AAT CA-3/)17.\n\nAmplification reactions for both genes were carried out in a total reaction volume of 10μL. The 10 μL PCR reaction mixes included 1 X PCR buffer, 5.0 mM MgCl2, 0.2 μM of each primer, 0.4 μL of 0.2 units of Taq polymerase, 0.25 mM of mixed dNTPs and 100ng of DNA template. The thermal profiles used were as follows: for 16S rRNA gene, initial step at 94°C for 5 minutes followed by 35 cycles of 94°C for 30 s, 53.9°C for 40 s and 72°C for 45 s, and a final step at 72°C for 5 min. For the COI gene, an initial denaturation at 94°C for 5 minutes, 35 cycles of 94°C for 45 seconds, 60°C for 45 seconds and a final step at 72°C for 1 minute, and concluded with a final elongation step at 72°C for 8 minutes followed by a hold at 4°C. PCR products were visualized on a 2% agarose gel stained with ethidium bromide and the most intense products were selected for sequencing. Purified DNA products were labelled using BIG Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems Inc., CA, USA) with ABI 3130Xl BigDye® Terminator model following manufacturer’s instructions. The PCR sequencing protocol cycling conditions were as follows: an initial step of 2 minutes at 96°C and 35 cycles of 30 s at 96°C, 15 s at 55°C, and 4 minutes at 60°C.\n\nIn order to assure the quality and integrity of the fish samples barcoded in this study, all the PCR amplified products and their corresponding DNA sequences were larger than 600 bp. This ensures that no nuclear DNA sequences originating from mt DNA sequences (NUMTs) being amplified as the limit of NUMTs rarely reach 600 bp. Standard nucleotide BLAST (BLASTN)18 and BOLD Identification System were used to compare the sequences and those sequences showing 99–100% alignment with no gaps or indels (insertions/deletions) was selected. The emphasis of these tools is to align regions of sequence similarity with the partial coding sequence of fish mitochondrial COI gene. The sequences for all the specimens were aligned using Clustal W as implemented in MEGA (version 5.2)19.\n\nThe total dataset (32) included 16 COI sequences and 16 16S rRNA sequences for 3 fish species comprising 16 individuals. The sequence similarity search for species identification was done in two public databases, viz., BOLD (http://www.boldsystems.org/index.php/IDS_OpenIdEngine) and GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The highest percent pairwise identity for each sequence blasted (BLASTN ) at NCBI was compared with the percent similarity scores of the same sequence within the BOLD-IDS (BOLD Identification System)20. Kimura 2-parameter (K2P) congeneric and conspecific variation21, neighbour joining (NJ) and maximum likelihood trees construction were done using the computer program MEGA Version 5.222, exported in newick format into FigTree version 1.4.223 for visualization and annotation.\n\n\nResults\n\nThe read length of COI and 16S rRNA gene sequences obtained were around 681 and 570 bp long, respectively. A total of 48 products were successfully sequenced for both sets of primers (Table 1). COI gene was sequenced bidirectionally using FishF1 and FishR1 primers while the 16S rRNA gene was sequenced only with the forward primer.\n\nThe three fish species sequenced were C. gariepinus, C. zillii and S. melanotheron, the size of each fish sequence obtained (all ≥ 500 bp) was in line with the BOLD-IDS prescription. Only 3 of the 32 samples analysed failed to yield a DNA barcode. All pseudogenes or contaminant sequences were deleted before analysing the sequences. A total of 696 nucleotide sites for the COI gene, and 1049 nucleotide sites for the 16S rRNA gene were observed. Using MEGA 5.2, analysis and exploration of the COI aligned sequences were computed: 347 sites are conserved, 346 are variable (polymorphic) and 225 are parsimony informative. Nucleotide composition analysis revealed the mean frequencies for the complete dataset to be 29.0% for T, 26.6% for C, 26.4% for A and 18.0% for G. The highest percentage G-C at 49.6% was detected in C. zilli, while the lowest 42.2% was in C. gariepinus. COI sequences contain 347 conserved sites out of 696 (49.86%) bp, 346 variable sites out of 696 (49.71%) bp, 225 parsimony informative sites out of 696 (32.33%) bp and 121 singleton sites out of 696 (17.39%) bp. Nucleotide composition of the 16S rRNA analysis gave a total of 1049 nucleotide sites and revealed the mean frequencies for the dataset to be 269 bp/site (25.64%) conserved, 338 (32.22%) variable, 183 (17.45%) parsimony informative and 155 (14.78%) singletons. The mean frequencies for the complete data were 31.2.0% for T, 22.3% for C, 22.5% for A and 24.0% for G.\n\nTable 3 shows the average number of identical pairs (ii) for COI as 313.33 of which the 1st, 2nd and 3rd codons were 556, 206 and 178 respectively. Transitional pairs (si) were found to be lower (si = 25) than transversional pairs (sv = 34). Ratio of si/sv (R) was 0.79 for the dataset. The average number of identical pairs (ii) for 16S rRNA was 153 of which the 1st, 2nd and 3rd codons were 154, 152 and 153 respectively. Unlike COI, transversion was the most common substitution detected for all 16S rDNA analysed. In contrast, it was only the transitional pair that was highest in the third codon position whereas transversional pairs were highest at the second codon position (14 and 16 for si and sv, respectively). The average ratio of si/sv (R) was 0.88 for the dataset.\n\nAll frequencies are average (rounded) over all taxa. ii = identical pairs, Si = transitional pairs, Sv = tranversional pairs, R = Si/Sv and Avg = average.\n\nUsing sequences obtained from the 16 fishes, genetic distances were calculated and compared among the 3 studied species. Table 4 presents the genetic intraspecific variation, which shows that the highest nucleotide divergence was observed in C. zilli with nucleotide diversity within the population (π) =0.184 for COI gene, while S. melanotheron had the lowest divergence with π=0.065. The highest divergence for 16S rRNA was observed in C. gariepinus with π=0.102, while the lowest was T. zilli with π=0.019.\n\nN: the number of sequences; Pi: nucleotide diversity within the population; H: number of different sequences types; K: average number of nucleotide differences within the population.\n\nThe estimated pairwise genetic distances based on Kimura 2-Parameter Model are presented in Table 3 and Table 6. The lowest nucleotide variation for COI (Table 5) of 0.17 was observed between S. melanotheron and C. zillii suggesting a close relationship between these two taxonomic forms. The highest percentage of sequence divergence of 0.49 was found between the C. gariepinus and C. zillii. The lowest nucleotide variation for 16 S rRNA (Table 6) of 0.06 (interspecies distance) was observed between S. melanotheron and C. zillii suggesting a close relationship between these two taxonomic forms. The highest percentage of sequence divergence of 0.61 was found between C. gariepinus and S. melanotheron.\n\nPairwise conspecific divergence was denoted by the number of base substitutions per site between species (below diagonal) with their corresponding standard error estimate(s) (above the diagonal). Complete deletion of all codon positions (1st, 2nd, 3rd, and Noncoding), were employed in this analysis. All positions. *Genetic distance resulting from intraspecific variation between C. gariepinus - S. melanotheron and C. zillii – S. melanotheron. Mean conspecific divergence, (MCD).\n\nPairwise conspecific divergence was denoted by the number of base substitutions per site between species (below diagonal) with their corresponding standard error estimate(s) (above the diagonal). Completed deletion of all codon positions (1st, 2nd, 3rd and Noncoding), gaps and missing data were eliminated and were employed in this analysis. All positions. *Genetic distance resulting from intraspecific variation between C. gariepinus - S. melanotheron and C. zillii – S. melanotheron. Mean conspecific divergence, (MCD).\n\nComparison of each barcode to the reference sequences submitted previously to BOLD and GenBank resulted in straightforward identification of three species that showed significant species specific similarities based on GenBank and BOLD databases. These databases revealed definitive identity matches in the range of 96%–100% for COI consensus sequences of the three studied species. BLAST results from BOLD database were in agreement with GenBank results in identification of these species, yielding between 99% – 100% identities, except for one sample of C. zillii, which had 86% maximum identity in GenBank and no match, which was garnered from BOLD-IDS.\n\nThe majority of the GenBank-based identification for all species yielded an alignment E-value of 0.0. GenBank results for C. gariepinus ranged between 99% to 100% identity whereas for S. melanotheron, the hits were precisely 99% similarity. In the same vein, BOLD-IDS returned hits in the range of 97.84% to 100% species similarity. The database accession numbers and percentage similarity reference sequences with significant species specific similarity obtained from GenBank for all C. gariepinus is as follows: APOO432.1 (98%), JQ699203.1 (99%), JQ699203.1 (99%), GU701827.1 (100%), JF894132.1 (99%), HM882821.1 (100%), AP012010.1 (100%), AP012010.1 (100%), AP012010.1 (99%). It also showed significant non-specific similarity (98%) for C. gariepinus (query) with Polypterus seneqalus (database accession number APOO432.1).\n\nOf the three C. zillii individual species barcoded in this study, significant species specific similarity was recorded for two individuals at 99% (GenBank) with accession numbers FJ348137.1 and an insignificant species specific similarity (86%) for one of the species with database accession number JX173760.1. BOLD-IDS also gave significant species specific similarity 99.44% and 99.65% for the C. zillii species and no match for one of the samples. These species also showed moderate species specific similarity at 93% and 96% with accession numbers HM882922.1 and HM882911.1 respectively and an insignificant species specific similarity (83%) for one of the species with database accession number HM882922.1.\n\nThus, when representative COI sequences for the 15 species were compared with existing data, 2 (13.3% of species) shared 100% identity with existing GenBank database entries, 12 (80.0% of species) shared 99% and just one product shared < 97% similarity. Thus, the studied species showed non-ambiguous match categories. GenBank database revealed moderate to definitive identity matches in the range of 93%–99% for consensus sequences of the three studied species with an E-value of zero for all samples. Unlike the COI gene, the GenBank database for C. gariepinus samples revealed definitive matches at 99% for all studied species except three sample out of which two samples 1 and 5 (C. gariepinus) were moderately species specific at 93% and 95% similarity, while sample 11 (C. gariepinus) was insignificant at 81% similarity. The accession numbers for all obtained C. gariepinus reference sequences are given thus, AP012010.1 (95%), JQ699188.1, JQ699187.1, JQ699184.1 and JQ699185.1 were all (95%) and Q699184.1, JQ699186.1, JQ699184.1, JQ699188.1, JQ699187.1, JQ699185.1 all (99%). For S. melanotheron, the percent similarities were species specific significant at 99% for the two species considered in this study with accession number GQ167976.1. It is worth mentioning that it also showed significant non-specific similarity (98%) for C. gariepinus (query) with P. seneqalus (accession number APOO432.1).\n\nThus, when representative 16S rRNA sequences for the 16 species were compared with existing data, 13 (81.25% of species) shared 99% identity with existing GenBank database entries, and 3 (18.75% of species) shared < 97% similarity. Two ambiguous or incorrect identifications represented by P. obscura were detected and were not included in the final data analysis in MEGA 5.2. Results obtained from similarity search of GenBank confirmed definite species identity for the three studied species but not all the individuals of the two species namely C. gariepinus and C. zillii produce a significant species specific similarity.\n\nThe evolutionary history was inferred using the maximum likelihood (ML) and neighbour joining (NJ) methods based on the Tamura-Nei model and number of difference models, respectively. A full K2P model-based NJ cladogram shows the genetic distance between all specimens that generated a DNA barcode as described above to provide an overview of sequence divergences between all species tested in this study. The consensus tree results computed by the NJ and ML methods are shown in Figure 1 and Figure 2. The distances estimated by the two methods were very similar and the preliminary test with these models built up similar topologies. According to the NJ tree computed for COI sequences (Figure 1B), the species in the present study were clustered independently within their corresponding genera. This means that closer species in terms of genetic divergence were clustered at the same nodes; However, C. gariepinus splits into two clades irrespective of the location. Interestingly, the family Cichlidae did not form an assemblage by clustering together, however, clustered separately within their genera before merging with a 100% bootstrap value. This result is similar to the ML tree obtained to confirm the COI sequence divergence. Moreover, the phylogenetic tree constructed with maximum likelihood method also shows a similar result to the NJ tree (Figure 1A).\n\nPhylogenetic analysis A. Maximum likelihood tree, constructed based on Tamura Neil 3P substitution model. B. Neighbour-joining trees based on number of difference model, constructed from COI gene sequences (Bootstrap test was 1000 replicates).\n\nPhylogenetic analysis A. Maximum likelihood tree based on Tamura Neil 3P constructed for 16S rRNA gene sequences. B. Neighbour-joining trees based on number of difference model constructed from16S rRNA gene sequences. (Bootstrap test was 1000 replicates).\n\nThe ML tree based on Tamura Niel 3P was computed for 16S and is presented in Figure 2A. Specimens of the same species did not cluster together as expected. Taxonomic deviation was detected at the species level for all the three studied species and these deviations were reflected at higher levels (genus and family) particularly in the ML tree. Although C. zillii is a member of Cichlidae, specimens were clustered separately with species of C. gariepinus and S. melanotheron, under separate nodes; however, the low bootstrap values in the upper portion of the tree suggest that the topology of the consensus tree is unreliable. The reverse was the case as indicated in Figure 2B, a close inspection of the K2P NJ tree revealed that distinction existed between two cichlids S. melanotheron and C. zillii relative to the ML tree. These species clustered separately within their own genera and were unambiguously separated. In other words, all specimens of the same species were clustered together. This also applied to C. gariepinus, although in each of these cases, 10 samples of C. gariepinus were clearly separated at the species level but under two different clades. They clustered under the same family and formed two separate clusters on the NJ tree.\n\n\nDiscussion\n\nThe DNA barcode approach provides additional important data for the precise identification and classification of diverse biodiversity24. The COI and 16S rRNA markers used in our study were useful for the identification of the studied fish species. Our observations in this study show that phylogenetically, COI sequences effectively clustered most conspecific and congeneric species. This was also observed in similar studies in Australian fishes25, Canadian freshwater fishes26, freshwater fishes from southeastern Nigeria13,14, Indian catfishes27 and freshwater fishes from Indonesia28. Earlier studies have described the utility of COI and 16S rRNA as candidate DNA markers29. Our results show that mtDNA COI has a higher degree of DNA variability than mt 16S rRNA and importantly, is widely used for fish identification30. Furthermore, recent studies have revealed that the 16S rRNA failed to distinguish closely related species due to their lower genetic variability31. We find that using the ML tree and the genetic pairwise distance matrix, the 16S rRNA failed to distinguish conspecific species in the sampled Tilapia populations, as shown by low bootstrap values in the phylogenetic tree. The low power of 16S rRNA can be attributed to the paucity of informative sites compared with mtDNA COI. Cawthorn and co-workers using the 16S rRNA were also not able to distinguish 53 commercial fish species31. However, species identification based on mtDNA COI was unambiguous, our results suggest that COI sequence provides sufficient genetic variability for all studied species especially sampled populations of C. zillii and S. melanotheron.\n\nThe mean genetic K2P genetic distances of COI were similar between intraspecific and interspecific species but different at the confamilial taxonomic level suggesting the absence of a barcoding gap, an observation also made by Zou et al.,32. A study on publicly available sequences of marine and freshwater fishes available from the Barcoding of Life Database also reported a paucity of barcoding gap in COI1,20. The cichlid populations showed varying degrees of introgression and hybridization with respect to the clariid family examined in this study. An explanation for this may be that the infrequent mating of closely related species may bring about hybridization of offspring’s, which for maternally inherited mitochondrial genes, may result in phylogenetic paraphyly33–35. There have been several reports of autochthonous hybridization between closely related species occasioned by human-induced-changes to local habitats. This may suggest parapatric speciation between C. zilli and T. guineensis in many rivers where they co-exist36. Most of the comparisons done in this study were within the 3% score mark and are in line with the suggestion of Wong and Hanner37, except for one species of sampled C. zillii, which returned no match. Thus, it was described further as unambiguous as BOLD-IDs suggested that it could either be C. zillii or T. guineensis. Within a conspecific distance of less than 2%, BOLD-IDS validates its identification search of a species query sequence. This is usually only when the species in the BOLD-IDS database contains at the least, three barcoded specimens22. Low (86%) match was also recorded with C. zillii sequences in GenBank. Thus, it is strongly suspected that this result is insignificant and that C. zillii sequences stored in both the BOLD and GenBank databases were originally specimens of T. guineensis or hybridized. Therefore, it can be interpreted that T. guineensis may be actually C. zillii with regards to NCBI BLASTN search.\n\nThe hallmark of barcode analysis is to delineate species boundaries. This is in conformance with our observed results utilising the NJ trees, as there was an obvious phylogenetic signal in COI sequence data38. All NJ trees for both markers resolved species-specific clades that were supported by moderate to high bootstrap values. C. gariepinus did not form a distinct clade even when they clustered together in both COI (NJ and ML trees) and 16S rDNA (only NJ tree). The clustering of C. gariepinus (Figure 1A) and C. gariepinus (Figure 1B) in different lineages is due to phylogenic separation. This has been confirmed in a similar study carried out by Funk and Omland35 and is congruent with the data derived from this study. In their study, geographical separation during early stages of their evolution resulted in C. macrocephalus and C. batrachus in one lineage and C. gariepinus in another lineage; C. macrocephalus and C. batrachus are native Asian catfish while C. gariepinus is of African origin. Geographic differentiation is therefore apparent for the C. gariepinus species, with one clade comprising the Oodoba (O) individuals and another comprising the Asejire (A) individuals. Our observations from results from the K2P/NJ tree reveal a clustering together of similar species. These results are in line with present taxonomic classifications of the three fishes studied.\n\nAnother anomalous observation in the ML tree of the 16S rRNA is that of S. melanotheron clustered with species C. zillii. This indicates deviations at the genus level and may indicate a shared haplotype although the bootstrap values obtained were significant. This aberration was not noticed with the COI as it clustered S. melanotheron specimens together. Therefore, the species did not cluster independently within their corresponding genera. The monophyly of exhibited by COI for the two cichlids was supported by 16S rRNA, but the position of the clade formed by C. zillii in relationship with S. melanotheron is dependent on the molecular marker selected for the phylogenetic analysis. The data reveal a close relationship between C. zillii and the clade formed by S. melanotheron, which is not surprising taking into consideration that they are both from the same family. The NJ method, which differentiated the 16S rRNA sequences without ambiguity compared with the ML tree, which failed to produce similar result and clustered the species erroneously indicating that they possess a shared haplotype. Thus, the 16S tree generated by the ML tree method was unable to separate the nucleotide sequences of three studied species. Consequently, this ambiguity can be resolved using more markers such as microsatellites, which have proved useful in species delimitation39.\n\nThe report emanating from this study was not wholly congruent with the phenomenon of increasing evolutionary divergences in taxonomic levels within and among species. This is supported by a case study carried out by Zou et al.,33 on Neogastropoda where the barcoding gap between levels of intraspecific variation and interspecific divergence does not exist in either analysis of COI or 16S rDNA sequences. This could be attributed probably to very limited sample sizes employed in this study at each taxonomic level28. However, despite this obvious limitation, one interesting observation is the genetic relatedness between S. melanotheron and C. zillii. The Tilapiine fishes, S. melanotheron and C. zillii, could not be separated by the 16S marker. This was because it lacked resolution in species differentiation, a key weakness of the marker. This was despite similar mean K2P-distances obtained at multiple taxonomic levels suggesting the lack of a barcoding gap. However, COI was able to clearly separate the fish species ruling out introgression of the species as responsible for this limitation. Our observations demonstrate the need for precise species identification in the generation of any barcode library. Thus, classification of both Clariid and Cichlid species from South West Nigeria has benefited from phylogenetic analysis using mitochondrial ribosomal genes as markers.\n\n\nConclusion\n\nAlthough Nigeria is not a major fishing nation, focus on freshwater and marine conservation, and studies about the early life history of all fishes are essential for the management of its aquatic resources. Therefore, identification of fishes based on morphological characteristics should be complemented with molecular methods of identification. In addition, other markers such as nuclear DNA can be used in identification and estimating fish population. Our results clearly underlie the efficiency of DNA barcoding in the identification of the three species in Southwest Nigeria. Proper species identification is important in fish conservation and management. Thus, DNA barcoding will be a useful tool for monitoring of conservation in fisheries management programmes in Nigeria.\n\n\nData availability\n\nRaw sequence data for the samples reported here can be found in GenBank under accession numbers: KX231778, KX231779, KX231780, KX231781, KX231782, KX231783, KX231784, KX231785, KX231786, KX231787, KX231788, KX231789, KX231790, KX231791, KX231792, KX231793, KX243276, KX243277, KX243278, KX243279, KX243280, KX243281, KX243282, KX243283, KX243284, KX243285, KX243286, KX243287, KX243288, KX243289, KX243290, KX243291.",
"appendix": "Author contributions\n\n\n\nM.F. and A.O. Designed study. M.F., A.O and B.O. analysed data. M.F. and A.O. performed experiments. All authors contributed towards writing the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nWard RD, Hanner R, Hebert PD: The campaign to DNA barcode all fishes, FISH-BOL. J Fish Biol. 2009; 74(2): 329–56. PubMed Abstract | Publisher Full Text\n\nBegg GA, Waldman JR: An holistic approach to fish stock identification. Fish Res. 1999; 43(1–3): 35–44. Publisher Full Text\n\nCaddy JF, Garibaldi L: Apparent changes in the trophic composition of world marine harvests: the perspective from the FAO capture database. Ocean Coast Manag. 2000; 43(8–9): 615–55. Publisher Full Text\n\nNunan F: Wealth and welfare? Can fisheries management succeed in achieving multiple objectives? A case study of Lake Victoria, East Africa. Fish and Fisheries. 2014; 15(1): 134–50. Publisher Full Text\n\nPimm SL, Jenkins CN, Abell R, et al.: The biodiversity of species and their rates of extinction, distribution, and protection. Science. 2014; 344(6187): 1246752. PubMed Abstract | Publisher Full Text\n\nSchroeder KL, Martin FN, de Cock AW, et al.: Molecular detection and quantification of Pythium species: evolving taxonomy, new tools, and challenges. Plant Dis. 2013; 97(1): 4–20. Publisher Full Text\n\nTautz D, Arctander P, Minelli A, et al.: DNA points the way ahead in taxonomy. Nature. 2002; 418(6897): 479. PubMed Abstract | Publisher Full Text\n\nJenkins C, Chapman TA, Micallef JL, et al.: Molecular Techniques for the Detection and Differentiation of Host and Parasitoid Species and the Implications for Fruit Fly Management. Insects. 2012; 3(3): 763–88. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchwarzfeld MD, Sperling FA: Comparison of five methods for delimitating species in Ophion Fabricius, a diverse genus of parasitoid wasps (Hymenoptera, Ichneumonidae). Mol Phylogenet Evol. 2015; 93: 234–48. PubMed Abstract | Publisher Full Text\n\nChiu TH, Kuo CW, Lin HC, et al.: Genetic diversity of ivory shell (Babylonia areolata) in Taiwan and identification of species using DNA-based assays. Food Control. 2015; 48: 108–16. Publisher Full Text\n\nRatnasingham S: BOLD Barcode of Life Data System, Version 3.2013.\n\nMatthews WJ: Patterns in freshwater fish ecology. Springer Science & Business Media; 2012. Reference Source\n\nNwani CD, Becker S, Braid HE, et al.: DNA barcoding discriminates freshwater fishes from southeastern Nigeria and provides river system-level phylogeographic resolution within some species. Mitochondrial DNA. 2011; 22(Suppl 1): 43–51. PubMed Abstract | Publisher Full Text\n\nNwakanma C, Ude G, Unachukwu MN: The Use of DNA Barcoding in Identification of Genetic Diversity of Fish in Ugwu-Omu Nike River in Enugu. Nigerian Journal of Biotechnology. 2015; 29(1): 27–33. Reference Source\n\nOlaosebikan BD, Raji A: Field guide to Nigerian freshwater fishes. 2nd Edn., Federal College of Freshwater Fisheries Technology, New Bussa, Nigeria, 2004; 111.\n\nBekele J, Hussien D: Prevalence of Internal Parasites of Oreochromis niloticus and Clarias gariepinus Fish Species in Lake Ziway, Ethiopia. Journal of Aquaculture Research & Development. 2015; 6(2): 1. Publisher Full Text\n\nPalumbi SR: Nucleic acids II: the polymerase chain reaction. In ‘Molecular Systematics’. (Eds. DM Hillis, C. Moritz and BK Mable.). 205–247.\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\nThompson JD, Gibson TJ, Higgins DG: Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinformatics. 2002; Chapter 2: Unit 2.3. PubMed Abstract | Publisher Full Text\n\nRatnasingham S, Hebert PD: bold: The Barcode of Life Data System (http://www.barcodinglife.org). Mol Ecol Notes. 2007; 7(3): 355–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980; 16(2): 111–20. PubMed Abstract | Publisher Full Text\n\nTamura K, Peterson D, Peterson N, et al.: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011; 28(10): 2731–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRambaut A: FigTree v. 1.4. 0. 2012. Reference Source\n\nBucklin A, Steinke D, Blanco-Bercial L: DNA barcoding of marine metazoa. Ann Rev Mar Sci. 2011; 3: 471–508. PubMed Abstract | Publisher Full Text\n\nWard RD, Zemlak TS, Innes BH, et al.: DNA barcoding Australia's fish species. Philos Trans R Soc Lond B Biol Sci. 2005; 360(1462): 1847–57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHubert N, Hanner R, Holm E, et al.: Identifying Canadian freshwater fishes through DNA barcodes. PLoS One. 2008; 3(6): e2490. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBhattacharjee MJ, Laskar BA, Dhar B, et al.: Identification and re-evaluation of freshwater catfishes through DNA barcoding. PLoS One. 2012; 7(11): e49950. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMuchlisin ZA, Thomy Z, Fadli N, et al.: DNA barcoding of freshwater fishes from lake Laut Tawar, Aceh Province, Indonesia. Acta Ichthyol Piscat. 2013; 43(1): 21–9. Publisher Full Text\n\nXia Y, Gu HF, Peng R, et al.: COI is better than 16S rRNA for DNA barcoding Asiatic salamanders (Amphibia: Caudata: Hynobiidae). Mol Ecol Resour. 2012; 12(1): 48–56. PubMed Abstract | Publisher Full Text\n\nZhang J, Hanner R: Molecular approach to the identification of fish in the South China Sea. PLoS One. 2012; 7(2): e30621. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChatellier S, Mugnier N, Allard F, et al.: Comparison of two approaches for the classification of 16S rRNA gene sequences. J Med Microbiol. 2014; 63(Pt 10): 1311–5. PubMed Abstract | Publisher Full Text\n\nCawthorn DM, Steinman HA, Witthuhn RC: Evaluation of the 16S and 12S rRNA genes as universal markers for the identification of commercial fish species in South Africa. Gene. 2012; 491(1): 40–8. PubMed Abstract | Publisher Full Text\n\nZou S, Li Q, Kong L, et al.: Comparing the usefulness of distance, monophyly and character-based DNA barcoding methods in species identification: a case study of neogastropoda. PLoS One. 2011; 6(10): e26619. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarraclough TG, Nee S: Phylogenetics and speciation. Trends Ecol Evol. 2001; 16(7): 391–9. PubMed Abstract | Publisher Full Text\n\nFunk DJ, Omland KE: Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annu Rev Ecol Evol Syst. 2003; 34: 397–423. Publisher Full Text\n\nPouyaud L, Agnèse JF: Phylogenetic relationships between 21 species of three tilapiine genera Tilapia, Sarotherodon and Oreochromis using allozyme data. J Fish Biol. 1995; 47(1): 26–38. Publisher Full Text\n\nWong EH, Hanner RH: DNA barcoding detects market substitution in North American seafood. Food Res Int. 2008; 41(8): 828–37. Publisher Full Text\n\nBJ Moniz M: Diversity, species concepts and phylogenetic relationships of some marine algae in a perspective of Biodiscovery (Doctoral dissertation). 2013. Reference Source\n\nVanhaecke D, Garcia de Leaniz C, Gajardo G, et al.: DNA Barcoding and Microsatellites Help Species Delimitation and Hybrid Identification in Endangered Galaxiid Fishes. PLoS One. 2012; 7(3): e32939. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "14760",
"date": "06 Jul 2016",
"name": "Dora Davies",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 contribute to the knowledge of species of freshwater fish of interest in Nigeria, whose diversity is poorly known, to using molecular tools that complement the difficult morphological studies by the shortage of taxonomists and lack of reliable morphological characters for discriminating species.\nConfirm the usefulness of COI, especially, to discriminate phylogenetically close taxa, although there is some overlap between the intraspecific and interspecific variation, which partially obscures the delimitation of species. The number of samples involves a remarkable effort, aimed at improving the accuracy of the results.\nMinor suggestions are given:\n\nIn the expression \"Tilapia (Coptodon)\" Coptodon seems a subgenre of Tilapia. One could say “Coptodon, previously included in Tilapia”\n\nIn the third paragraph of the introduction, second line, should write the name of the genus Clarias and not its abbreviation, C., because it could be confused with the name of the other gender that also begins with C, Coptodon.\n\nIn the second paragraph, second line of PCR and DNA sequencing item, add a space between 10 and μL after point.\n\nIn the body of Table 1 labels the fifth row are not necessary. You should delete the row.\n\nTable 4 does not include data for H: number of different types sequences; K: average number of nucleotide differences within the population. Add the data in the table.\n\nIn the paragraphs where the authors compare the sequences of COI and 16S rRNA with those published in GenBank, the term species use instead of specimens. Then replace species by specimens in all cases.\n\nFigure 1 and 2: numbers are unclear and are above the lines.",
"responses": []
},
{
"id": "16270",
"date": "29 Nov 2016",
"name": "Shadi Shokralla",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe current MS is a good case study showing the efficiency of the DNA barcoding in study the diversity of different groups of organisms.\nI have some comments to improve the MS:\nThe sample size is very small to conduct any diversity measure.\n\nAll gene names should be italic.\n\nHow many mg was the starting material for DNA extraction?\n\nI was expecting to see more taxonomic identification in this work. Also it will be great if the authors can add some information about the habitation of these tested species.\n\nI suggest to the author to do some phylogenetic comparisons between CO1 and 16S not only with the species tested in this work but also other species of the same genus or other species of the same family to enrich the study more.",
"responses": []
}
] | 1
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https://f1000research.com/articles/5-1268
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https://f1000research.com/articles/5-1261/v1
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07 Jun 16
|
{
"type": "Research Article",
"title": "What does the UK public want from academic science communication?",
"authors": [
"James Redfern",
"Sam Illingworth",
"Joanna Verran",
"Sam Illingworth",
"Joanna Verran"
],
"abstract": "The overall aim of public academic science communication is to engage a non-scientist with a particular field of science and/or research topic, often driven by the expertise of the academic. An e-survey was designed to provide insight into respondent’s current and future engagement with science communication activities. Respondents provided a wide range of ideas and concerns as to the ‘common practice’ of academic science communication, and whilst they support some of these popular approaches (such as open-door events and science festivals), there are alternatives that may enable wider engagement. Suggestions of internet-based approaches and digital media were strongly encouraged, and although respondents found merits in methods such as science festivals, limitations such as geography, time and topic of interest were a barrier to engagement for some. Academics and scientists need to think carefully about how they plan their science communication activities and carry out evaluations, including considering the point of view of the public, as although defaulting to hands-on open door events at their university may seem like the expected standard, it may not be the best way to reach the intended audience.",
"keywords": [
"Science communication",
"public engagement",
"outreach"
],
"content": "Introduction\n\nEngaging members of the lay public with science has become a business-as-usual activity for many scientists within academia. Although a widely accepted definition is yet to exist, science communication can be considered an umbrella term for public engagement, outreach, widening participation etc., with the specifics depending on the mode of delivery, aim, location, intended audience, and institutional preference1. Common examples of science communication include university open door events, science festivals, school visits, writing for the popular press, providing engaging presentations in easily-accessible locations (pubs, cafes etc.), using social media, and producing digital media such as podcasts and video content. The overall aim in all cases is primarily to engage a non-scientist with a particular field of science and/or research topic, often driven by the expertise of the academic.\n\nSupport for these science communication activities continues to grow following a pivotal report by the Wellcome Trust and the Office of Science and Technology in the UK, which recommended moving away from a deficit model2, whereby scientists assume the public have a lesser knowledge of science and provide information to fill any gaps. The deficit model has been discussed and criticized widely by many [for example 3–5], with science communicators favouring a contextual approach to establish dialogue between the experts and the members of the general public. This approach has been incorporated into the ethos and policy of many leading public engagement organisations such as the National Coordinating Centre for Public Engagement (NCCPE), which defined public engagement as “…a two-way process, involving interaction and listening, with the goal of generating mutual benefit.”6.\n\nThe UK Research Council (RCUK) representing all of the research councils in the UK, and many other research funders (e.g. The Wellcome Trust) require all applicants to integrate science communication in their research plan. Additionally, the 2014 Research Excellence Framework (REF) exercise, whereby the UK Government assessed all university research output in order to distribute funding, included an assessment of research impact, which was often supported with evidence of effective science communication, thereby helping to establish this “as a core part of business, not just good intentions”7.\n\nIt is therefore no surprise that there is a growing trend within universities to encourage their academic staff to undertake activities that may be considered as science communication. Researchers have indicated many important motivations for these activities, including increasing the public’s interest of science and awareness as well as an appreciation of science and scientists8,9.\n\nScience festivals often encompass obvious outputs of academic public engagement. Described as a ‘celebration’ of science, technology, engineering and related aspects, with the intention to engage non-specialists with a time-limited recurring frequency, they are growing in number globally, with the UK hosting more than any other country10.\n\nWhilst the attraction and effect of science festivals on participating public are well researched [e.g. 11–13], there is little in the literature to suggest that this is the preferred method of communication for the audience (‘the public’), nor how members of the public would actually like to engage with academic scientists if they had the choice. Science festivals are of course not unique in this context, with many different forms of communique from ‘popular science’ books14 to blogs14,15 and podcasts15,16 often receiving support depending on the individual preferences of the author/presenter/scientist. Indeed, incidental evidence collected by the authors of this study suggest that the mode of delivery, methods, and most importantly the scientific topic of many science communication strategies are not decided in consultation with the general public, but are instead determined by constraints such as time, resources, skills and finance. This paper describes a preliminary small-scale study to assess what science communication output the public may like to see, and suggests how academics may be able to better engage with them as a result of this.\n\n\nMethod\n\nA questionnaire-based e-survey was designed with ten questions, four of which captured demographic details of the respondents (Table 1). The aim of the survey was to provide insight into respondent’s current and future engagement with science communication and/or public engagement activities. The intended respondents were anybody who did not identify as a scientist, therefore the only exlusion critieria was self-identification as a scientist.\n\nThe survey was made available for one month (May 2015) and advertised through the social media accounts of the authors (Twitter, Facebook and LinkedIn), with participants encouraged to share the survey with anybody who did not identify as a scientist. Ethical clearance was obtained prior to data collection from the Manchester Metropolitan University Ethics Board (document number SE141518), and carried out according to the British Educational Research Association’s (BERA) ethical guidelines for educational research; all responses were collected anonymously. Data were analysed using NVivo® (v10; QSR International Pty Ltd, Melbourne), where individual responses were coded into themes that were dertermined after an initial analyisis for the purpose of detailed analysis and discussion.\n\n\nRespondents\n\nA total of 112 responses were collected from respondents from ten countries. The nationalities of the participants were: United Kingdom (71.4%), United States of America (17%), Canada (2.7%), Netherlands (1.8%), Australia (1.8%), Germany (0.9%), Tanzania (0.9%), New Zealand (0.9%), Norway (0.9%), Spain (0.9%) with 0.9% not providing an answer. There were more male responses (56.3%) than female (42.9%) with 0.9% not providing an answer.\n\nThe most frequent (34.8%) age of respondents was between 20–29 years old, followed by 30–39 years old (20.5%), 50–59 years old (18.8%), 40–49 years old (12.5%), <19 years old (6.3%), 60+ years old (6.3%) and 0.9% not providing an answer. Respondents reported a wide range of educational backgrounds from no formal qualifications to doctoral degrees (Table 2). All respondents replied no when asked if they were a scientist.\n\n\nResults and discussion\n\nThe majority of respondents agreed that they were interested in science (87.5%), with only 7.1% stating the opposite. Of the remaining respondents, 4.5% selected ‘other’ and provided mixed opinions whilst 0.9% did not provide an answer. Respondents provided a range of comments, which were coded into 18 different themes (Figure 1). The most common reason (21%) given for interest in science attempts to understand the unknown.\n\n“It's based on facts, discoveries and inventions improve our lives, it's interesting to know how the world works”\n\nThe second and third most common themes were the ethos of science (scientific method and its basis in evidence-based practice), and the perception that science is beneficial to society (both 13%). One respondent had mixed views about these aspects, noting that whilst science allows people to discover the unknown, the ‘dogma’ of science and the inability to access scientific literature made it difficult for people to interact with. This is interesting and supportive to the ongoing Open Access movement within academia, attempting to make all peer-reviewed research manuscripts freely available to everyone. Other respondents specified favoured disciplines; biology (12.5%), physics (8.8%), chemistry (3.7%), environmental or earth sciences (1.5%), social sciences (1.5%), mathematics (0.7%) and sustainability studies (0.7%).\n\nOf the respondents who stated that they had no interest in science, no explanation related to a specific scientific discipline. Instead they referred to personal experience with science education (50%), the conflict scientific method can have with religion (25%), or a personal dislike of scientists themselves (25%).\n\n“Snobs, know it all! Better than others just because they are intelligent, boring and thinking everyone should know what they know!”\n\nOne respondent, when discussing their education, detailed how intrigued they often are, but are made to feel inadequate and lacking in capacity and understanding.\n\n“…like it’s no big deal, and my wonder and awe is ill inspired”.\n\nTo understand how respondents engaged with science in their day-to-day lives, they were asked to select either very regularly, somewhat regularly, somewhat infrequently or never (Figure 2). When asked about different engagement mechanisms, the most frequently selected mechanism was via the Internet, with 55% of respondents using it very regularly, and a further 25% somewhat regularly. A further four mechanisms were used by over 50% of respondents at least somewhat regularly, these were: non-specific websites such as news pages, social media (28% very regular, 30% somewhat regular), specific websites such as blogs (30% very regularly, 24% somewhat regularly), and television (16.8% very regularly, 36.2% somewhat regularly). Interestingly, the top four are all online methods of communication, and represent all of the online options provided. Academics too are actively engaged with social media to network, discuss, plan and carryout studies17, with 80–90% of research scientists being at least aware of Twitter and Facebook18. This rich and existing community of academics could therefore be utilised more frequently and imaginatively to engage members of the public.\n\nTraditional media, i.e. magazines (10% very regular and 21% somewhat regularly), newspapers (9% very regular and 17% somewhat regularly) and radio (6.25% very regular & 12.5% somewhat regularly) are used less often to access science. A small group of respondents used physical events, such as museum visits (8% very regularly and 20% somewhat regularly), or attendance at science festivals to engage (3.5% very regular and 7% somewhat regularly). As noted in the introduction, academics practising science communication and engagement appear to favour these types of activities for public engagement events19, perhaps due to opportunity to adhere to a more standard format they are comfortable with – e.g. standing in front of people and explaining.\n\nSome respondents noted other methods for engagement with science that were not included in the survey. They were: multimedia content (e.g. DVD and on-demand streaming services, n=3), amateur science (e.g. amateur astronomer club or designing experiments for their children, n=3), through their workplace/job (e.g. working in a library, n=2), and by reading (books and academic literature, n=2).\n\nRespondents were also asked which events/activities aimed at engaging the public with science, if any, they had previously attended or participated in. Of all the respondents who provided a 33.8% of them had not attended any science engagement events.\n\n“No, I've never been aware of such an event.”\n\nMuseums were the most popular attended location (23.5%), with 13.2% attending but not specifying details. Universities and science festivals/fairs had a similar response rate (11.8% and 10.3% respectively); only 1.5% had previously engaged in a science communication activity online. This contrasts with the responses from the previous question, which revealed that the Internet was used to actively engage with science (Figure 2). It seems that if the curiosity is driven by the consumer rather than prescribed by the academic, then engagement with science is better recognised. The role of the scientist in creating activities is of little concern.\n\nThe following questions asked respondents how scientists and other organisations such as museums and universities may encourage people to attend events to see/discuss/participate with science. Respondents provided a wide range of reasons (n=146) or actions that may alter their decision to attend a science communication/outreach event (Figure 3). The most common theme related to their interest in the topic (21.9%). Suggested topics were wide-ranging, from science of practical value to the respondent, to any topic that the respondent may find interesting as long as it followed scientific methodology.\n\n“Novel exhibitions (e.g. opportunity to try a jet pack)”\n\nAdvertising and visibility of events were also considered (13.7%), with many respondents suggesting that they rarely see science communication events being advertised, but generally would attend if they knew far enough in advance. Some respondents elaborated on this, suggesting that advertising should be carried out on social media (n=6) whilst two respondents suggested that advertising be written with inviting language, so as not to intimidate or patronise any potential attendees.\n\nAlso of concern was the ability to engage with a hands-on practical demonstration with “more than just storyboards” (13.7%) as well as the geographic location (8.9%) of the activities, as some respondents lived in locations away from major universities or other organisations and so did not have the option to attend without travelling. These responses provide an interesting consideration for scientists; whilst many may consider hands-on practical demonstrations of science to be a valuable tool for the effective communication of science (again, similar to experiences they may have within the undergraduate classroom), participation will be limited to those within the commutable area. Providing further support to develop science communication activities within the digital landscape may be one way to enable participation with people in any geographical location, and is a change that is likely to be embraced given previous answers discussed relating to Internet-based engagement (Figure 2).\n\nDespite the interesting opportunities for active involvement that citizen science and crowd sourcing can offer [e.g. 20], only two respondents specified they would like experiments they could investigate themselves. Interestingly, the lecture format was suggested to a lesser extent (3.4%), a low approval for an anecdotally common format. Other considerations included cost (notably the requirement for events to be free, 6.8%) and the inclusion of enthusiastic (2.7%), or famous (2.7%) scientists.\n\nRespondents were asked to consider how they themselves could be better engaged by scientists trying to communicate scientific information (Figure 4). The most common answer was to bring scientific information to social media (41%). Of these respondents, 34% specified a type of social media (Figure 5), the most common being blogs, followed by Twitter, Facebook and YouTube.\n\nn=106.\n\nNot all respondents who selected social media provided a specific answer. n=32.\n\nSuggestions for output content via social media were, interestingly varied. The need for “idiot proof websites” and short scientific “facts” to gain people’s attention, contrasted with suggestions for “more detailed” and “accurate” online dialogue, reflecting the two types of social media suggested, Twitter, where currently only short snippets of information can be posted at any given time due to 180 character limits; and blogs, where the norm is to create pieces of writing of variable length, allowing more detail. Within the comments regarding social media, there was an overall consensus that its convenient nature makes it an attractive mode for scientists and audiences to interact.\n\nThe second most common theme was open-door events, but only one respondent provided any detailed thought, suggesting open-door events should take place within a University. Two respondents linked hands-on activities with open-door events, but it may be assumed from previous responses that people associate hands-on activities with open-door events, and thus did not feel the need to discuss them separately. Events in ‘real life’ locations (outside of typical engagement locations e.g. university or museum) were also suggested (12.2%). Including Café Scientifique (http://cafescientifique.org) or The Green Man music festival (http://www.greenman.net/).\n\nRespondents also suggested that online resources or websites were useful (but not specifically social media, 11.3%). A slightly smaller percentage of respondents recommended podcasting as a medium to better engage them with science (8.5%). Debate, discussion and interviews were all mentioned with reference to podcasts. Other themes resulting from this question included providing open-access literature (3.8%), community-specific events (such as local astronomy clubs, 1.9%), non-fiction books on scientific topics, better news coverage of scientific topics and more famous scientist personalities (0.9%). Also, 0.9% of responses suggest scientists should continue as is, as they believed scientists were successfully achieving their aim in effectively communicating with the public.\n\nThe age at which engagement events should be aimed was also investigated. The majority of responses suggested events should be split equally between adult-oriented and child-oriented (60.2%). The number of respondents suggesting that events should be aimed at children, adults or teenagers alone were similar (12.5%, 9.8% and 9.8% respectively). A small number of respondents believed events should be designed for families as a whole (3.6%), whilst the same number felt that the level of pre-existing knowledge should be the definition of who an event is aimed at, not the person’s age.\n\n\nConclusions\n\nThis study has provided new insights into how populations within the ‘general public’ may wish to engage with academic scientists who are interested and/or required to undertake elements of science communication. Whilst the authors note a variation in responses, that is, no single answer, the results highlight the complexity of communicating with the public which academic scientists need to understand further to ensure outcomes are as effective as possible.\n\nThe potential use of the Internet and digital technologies seems favoured by respondents, with less of a focus on more ‘traditional’ academic science communication activities such as science festivals and open door events. However, the public still want access to these types of events, with considerations such as time, cost, geographical location, clear/accessible advertising (e.g. through social media) and a non-patronising approach should be undertaken. Another key point is that of targeting age groups, with the majority of respondents keen to see adults engaged just as much as children. The type of information the public believe they would be interested in varies, however the themes of understanding the unknown, and the benefits science brings to society were noticeably popular. In order for academic science communicators to undertake activities of this nature, training needs to be introduced/amended to include the point of view of the audience, considering other more accessible methods such as online delivery. This will inevitably not be true for all activities, and even where appropriate, new skills, technology and advertising strategies are likely to be required.\n\nThe outcomes of this paper should pose as food-for-thought for proponent science communicators, with more research needed to be done to better understand how different groups of the wider community can be better served by science communication activities presented in different formats. What is evident is the need to think carefully about how scientists plan their science communication activities and carry out evaluations, including considering the point of view of the public, as although defaulting to hands-on open door events at their university may seem like the expected standard, it may not be the best way to reach the intended audience.\n\n\nData availability\n\nF1000Research: Dataset 1. Survey responses, 10.5256/f1000research.8815.d12388121",
"appendix": "Author contributions\n\n\n\nJR conceived of the study, participated in design of the study, data collection, data analysis and drafted the manuscript. SI and JV participated in design of the study, data analysis and drafted the manuscript. All authors gave final approval 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\nIllingworth S, Redfern J, Millington S, et al.: What's in a Name? Exploring the Nomenclature of Science Communication in the UK [version 2; referees: 3 approved, 1 approved with reservations]. F1000Res. 2015; 4: 409. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOffice of Science and Technology and The Wellcome Trust: Science and the Public: A Review of Science Communication and Public Attitudes to Science in Britain. London: Office of Science and Technology and The Wellcome Trust, 2000. Reference Source\n\nMayhew MA, Hall MK: Science Communication in a Café Scientifique for High School Teens. Sci Commun. 2012; 34: 546–554. Publisher Full Text\n\nDavies S, McCallie E, Simonsson E, et al.: Discussing dialogue: perspectives on the value of science dialogue events that do not inform policy. Public Underst Sci. 2008; 18(3): 338–353. Publisher Full Text\n\nBesley JC, Tanner AH: What Science Communication Scholars Think About Training Scientists to Communicate. Sci Commun. 2011; 33(2): 239–263. Publisher Full Text\n\nNCCPE: What is public engagement? 2016; n.d. [cited 18/01/2016]. Reference Source\n\nNCCPE: After the REF - Taking Stock: summary of feedback: NCCPE. 2014. Reference Source\n\nMartín-Sempere MJ, Garzón-García B, Rey-Rocha J: Scientists' motivation to communicate science and technology to the public: surveying participants at the Madrid Science Fair. Public Underst Sci. 2008; 17(3): 349–367. Publisher Full Text\n\nBurchell K: Factors affecting public engagement by researchers. London: The Wellcome Trust. 2015. Reference Source\n\nBultitude K, McDonald D, Custead S: The Rise and Rise of Science Festivals: An international review of organised events to celebrate science. Int J Sci Educ, Part B. 2011; 1(2): 165–188. Publisher Full Text\n\nFogg-Rogers L, Bay JL, Burgess H, et al.: “Knowledge Is Power”: A Mixed-Methods Study Exploring Adult Audience Preferences for Engagement and Learning Formats Over 3 Years of a Health Science Festival. Sci Commun. 2015: 37(4): 419–451. Publisher Full Text\n\nJensen E, Buckley N: Why people attend science festivals: Interests, motivations and self-reported benefits of public engagement with research. Public Underst Sci. 2014; 23(5): 557–573. PubMed Abstract | Publisher Full Text\n\nBultitude K: Science festivals: Do they succeed in reaching beyond the 'already engaged'? J Com. 2014; 13(4). Reference Source\n\nTurney J: Popular science books. Handbook of public communication of science and technology. 2008; 5–14. Reference Source\n\nKouper I: Science blogs and public engagement with science: Practices, challenges, and opportunities. J Com. 2010; 9(1): 1–10. Reference Source\n\nBirch H, Weitkamp E: Podologues: conversations created by science podcasts. New media society. 2010; 12(6): 889–909. Publisher Full Text\n\nMollett A, Moran D, Dunleavy P: Using Twitter in university research, teaching and impact activities. 2011. Reference Source\n\nVan Noorden R: Online collaboration: Scientists and the social network. Nature. 2014; 512(7513): 126–129. PubMed Abstract | Publisher Full Text\n\nBurchell K, Franklin S, Holden K: Public Culture as Professional Science. London: BIOS, London School of Economics and Political Science. 2009. Reference Source\n\nDickinson JL, Shirk J, Bonter D, et al.: The current state of citizen science as a tool for ecological research and public engagement. Front Ecol Environ. 2012; 10(6): 291–297. Publisher Full Text\n\nRedfern J, Sam I, Joanna V: Dataset 1 in: What do people in the UK want from academic science communication? F1000Res. 2016. Data Source"
}
|
[
{
"id": "14241",
"date": "17 Jun 2016",
"name": "Laura Bowater",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 does indeed provide food-for-thought for proponent science communicators. There are a plethora of science communication definitions and modes of delivery and this paper raises the intriguing possibility that science communicators may be getting left behind as the online revolution continues to take place and evolve. Although I feel there are a couple of caveats, for example with the audience reached through the particular sampling technique employed by the authors, this raises intriguing questions about the move towards two-way engagement and the difficulties that this will raise if online or indirect modes of delivery appear to be most valued by the public or at least members of the public reached by these authors.\nI wonder if the authors would like to contextualise why they have chosen to talk about science festivals within the Introduction? It may also be of value to expand this section a tad. For example, authors could provide examples of science festivals within the UK and perhaps more globally. What does a typical science festival look like? How long does it last? Where does it take place? Who is invited?.. etc Also, perhaps the authors could describe why scientists choose this direct interaction as a medium? What are the advantages or disadvantages and is it a format that engenders two-way participatory engagement?\nAlso, as the results indicate a desire for indirect science communication methods through online material etc perhaps the authors could expand a little on these types of delivery in the introduction too.\nIn the introduction the authors state that:\n\"This paper describes a preliminary small-scale study to assess what science communication output the public may like to see, and suggests how academics may be able to better engage with them as a result of this.\"\nI would temper this slightly. I suggest to the authors that instead they state:\n\"This paper describes a preliminary small-scale study to assess which science communication outputs the public may like to see, and suggests how academics may be able to better engage with them, within their own personal constraints, as a result of this.\nWith the methods section: The questionnaire raises some interesting questions. Would the authors be prepared to share why they chose these particular questions? This may help others who may be considering undertaking similar research into other aspects of science communication.\nWith the results section: The authors state: \"The majority of respondents agreed that they were interested in science (87.5%), with only 7.1% stating the opposite.\" How does this compare to MORI surveys carried out every four years in the UK that asked similar questions? https://www.ipsos-mori.com/researchpublications/researcharchive/3357/Public-Attitudes-to-Science-2014.aspx\n\nFigure 1 I suggest replacing theme with themes.\nThe authors state that \"One respondent had mixed views about these aspects, noting that whilst science allows people to discover the unknown, the ‘dogma’ of science and the inability to access scientific literature made it difficult for people to interact with\", would they consider illustrating this with a quote from the material?\nThe authors suggest that interestingly, the top four are all online methods of communication, and represent all of the online options provided. Academics too are actively engaged with social media to network, discuss, plan and carry out studies, with 80–90% of research scientists being at least aware of Twitter and Facebook. This rich and existing community of academics could therefore be utilised more frequently and imaginatively to engage members of the public. This is an interesting comment and I know a lot of scientists would be glad to hear this. However I suggest that the authors temper this by stating...'in this study\". It is worth remembering that respondents to the survey were contacted via online/ social media and you may be getting the views of the online-literate members of society that might not reflect the wider non-social media society.\nI wonder about the concept hinted at in the introduction, that academics practising science communication and engagement appear to favour these types of activities for public engagement events. I think there are many scientists who are turning to online indirect methods of sci comm to share areas of personal and wider science interest. Do the authors know if anything has been written about this at all? As a keen online provider of science material and functioning within these type of communities I wonder if I am seeing the result of my own biases as I feel all scientists are equally engaged this way as the community is vibrant and active and perhaps masks a wider science community that doesn't engage in this way. I am also intrigued that if this appears to be a preferred way for non-scientists to engage with science what does that mean for two-way engagement?\nFinally \"Despite the interesting opportunities for active involvement that citizen science and crowd sourcing can offer\" As someone actively involved in a citizen science project myself, I am intrigued by this result. It is thought provoking and deserves further attention.\nThere are some interesting finding within this publication especially within the context of the deficit/fully engaged models that have framed the science communication discipline and community. I recommend that is is indexed.",
"responses": []
},
{
"id": "14242",
"date": "20 Jun 2016",
"name": "Massimo Caine",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 develops an interesting and very much debated aspect of science communication i.e. which means ought be adopted by scientists and science communicators in order to successfully reach the lay-audience. Given the steep rise of different sources of scientific information (inside and outside the academic community), it is a timely submission that provides an analysis of the different possible ways to reduce the distance between scientific researchers and broad public. Importantly, this publication offers valuable food-for-thought coming from a privileged point of view, the layperson, attempting to address the flaws that the deficit model in communication may pose. As a matter of fact, by using an ad-hoc survey, Redfern et al. investigate which are the expectations of a non-scientific audience in terms of their engagement in scientific discussions. Remarkably, even though science fairs, festivals, citizen projects are the mainstream in many academic communication policies, the output of the survey intriguingly indicates that, if requested to choose, the public would prefer to be engaged with “time-saver” and less constrained approach like what can be found within the online environment.\n\nThe authors have widely addressed the survey results, providing a perspective that should be carefully considered by academic policies, not only in terms of communication departments, but also in terms of scientists training. However, there are some minor points that, as a reader, I would be interested to see expanded and that the authors may wish to include in their final version:\nthe authors mention that science festivals that are more present in UK than in every other country - a brief description of some of them in terms of format and public-deliverables would be an asset for the introduction;\n\nin order to better contextualize the preference of being engaged on an online format rather than with a science festival (fairs, open doors, hands-on workshops, citizen projects), it would be useful to know how much the selected audience of the survey have had the possibility to be “immunized” with such kind of events. Specifically, is there any correlation between the geographical origin of the public reached by the authors and the presence (or not) of science festival within the region? This would allow the authors to define weather if the lack of interest in science fairs is due to geographical proximity (i.e. among the 112 selected participants, none or few of them lives close to the place where a festival has been taking place lately) or if it is an opinion generated by the comparison between the two way of engagement (festivals vs. online tools). In my opinion, this would clarify if festivals were not a preferred choice due to their non-commutable distance from the surveyed respondent.\n\nProvided that one of the preferred choices of engagement are website and blogs, a brief description of some of these available supports and the way they deliver the scientific information would be a plus\n\nTo conclude, I believe that, with this submission, Redfern et al. provide a seminal data-driven publication about the expectations that the broad public may have when being engaged with science. Even though, the sampling of the survey is made on a reduced scale (112 participants), it definitely poses the basis for further research which will push forward the methods used by the science communication community in order better reach the lay-audience in the fast-pace-changing and hyper-connected era that we are living in.\n\nI am looking forward to reading the final version of the manuscript and, in the meantime, I definitely recommend this article for being indexed.",
"responses": []
},
{
"id": "14240",
"date": "24 Jun 2016",
"name": "Helen L. Brown",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 analyses the results of a questionnaire completed by a group of non-scientists. The questions focused on how the participants currently engage with science and how they might like to do so in the future. Overall the paper provides a well written description of the study and highlights the key results. This paper will be of particular interest to those within the science community who are heavily engaged in promoting and/or carrying out pubic engagement events as it highlights that the current model of outreach which is favoured by many providers does not overlap with those activities favoured by participants in this study. This highlights that when designing events in order to engage and reach the widest possible audience the public should also be consulted.\nMinor comments\nIntroduction\nScience festivals are discussed in the introduction but there is little description of the other outreach methods/events that were mentioned within the questionnaire. It might be of use to also give examples of some of the other engagement methods.\nMethods\nit would be useful to apply some statistical analysis to the results, although I appreciate that statistical analysis may be limited by the sample size of the study.\n\nMajor comments\nMethod\nThe paper analyses a questionnaire which was distributed via the authors social media accounts. This method of distribution may significantly bias the results obtained. In particular I would expect respondents who had answered a questionnaire advertised in the digital environment to encourage more online engagement from scientists, which was found. This should be considered when discussing the results.\n\nSimilarly, although the authors refer to respondents as representing the 'general public' it could be argued that the respondents already have a enhanced level of scientific engagement, since they engage with the authors social media and chose to compete the questionnaire. This bias should also be addressed by the authors.\n\nOverall I think this is a worthwhile and thought provoking preliminary study and hope that the authors will continue the work in the future. I would be very interested to see if the same conclusions can be drawn from a wider participant group and to look in more detail about what engagement methods the public find the most worthwhile.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1261
|
https://f1000research.com/articles/5-1253/v1
|
07 Jun 16
|
{
"type": "Research Note",
"title": "Decomposition of mutational context signatures using quadratic programming methods",
"authors": [
"Andy G. Lynch"
],
"abstract": "Methods for inferring signatures of mutational contexts from large cancer sequencing data sets are invaluable for biological research, but impractical for clinical application where we require tools that decompose the context data for an individual into signatures. One such method has recently been published using an iterative linear modelling approach. A natural alternative places the problem within a quadratic programming framework and is presented here, where it is seen to offer advantages of speed and accuracy.",
"keywords": [
"mutational context",
"signatures",
"cancer aetiology"
],
"content": "Introduction\n\nThe existence of context-specific DNA mutational signatures as a response to carcinogens has been known for some time (see e.g. Pfeifer et al.1), but the last three years have seen progress to bioinformatic inference of mutational signatures from large scale cancer sequencing studies2–4 such as TCGA (http://cancergenome.nih.gov/) and ICGC (icgc.org).\n\nThese methods of signature discovery, while important, do not translate to clinical application. First of all, they are reliant on a large corpus of samples for their efficacy, making them impractical to be run repeatedly for each new patient. Secondly, even with a large corpus, the results for one individual can theoretically change depending on the identities of the other patients in the corpus, which is undesirable in practice. Therefore there is great value in methods such as those recently presented by Rosenthal et al.5 that can, for a single sample, break a vector of observed mutation counts into constituent signature components.\n\nIn the Cancer Research UK funded oesophageal adenocarcinoma ICGC project we have taken a similar view to Rosenthal et al.5 for the decomposition of a single sample, but rather than decomposing mutational contexts into signatures by fitting iterative linear models (ILM), we have viewed the question as lying within the framework of quadratic programming (QP). By mutational contexts, we commonly mean the 96 trinucleotide contexts consisting of the 6 distinguishable mutations and the 16 combinations of immediately preceding and following bases. More general definitions are possible3 and can be accommodated in both the QP and ILM approaches, but we assume the standard 96 in what follows.\n\n\nMethods\n\nIn brief, we want to minimize the difference between the normalized observed vector of mutation contexts m (a 96 × 1 vector) and Sw (where S is a 96 × k matrix, each column of which represents the contributions of mutational contexts to one signature, k is the number of known mutational signatures, and w is a k × 1 matrix of weights to be estimated). Our problem, then, is to:\n\n\n\nwhich is equivalent to:\n\n\n\nwhich is the classical quadratic programming problem that can be solved quickly (given the form of STS) and easily using the core linear algebra functionality of R (version 3.2.4)6 and the quadprog package (version 1.5-5)7, which implements the dual method of Goldfarb and Idnani8,9 to find the solution. Practical details of the implementation can be found in the ‘Data and Software Availability’ section of this note.\n\n\nResults\n\nIn most circumstances, both the ILM and QP approaches work well. Illustrating them on an example from the OCCAMS consortium’s whole-genome sequencing of oesophageal adenocarcinoma10, we see that the ILM and QP approaches are highly concordant (See Figure 1). The ILM approach has the advantages of familiarity of interpretation, and enforcement of parsimony should this be desired (while parsimony is generally desirable if building a predictor, if we are trying to model an underlying truth then it represents a strong assumption). More importantly, taking advantage of the linear modelling framework, it would be easy to generalize this approach to use other error models or to include additional structure should one e.g. wish to simultaneously investigate several related samples.\n\n18, 916 SNVs from sequencing library SS6003314 (tumour) compared to library SS6003313 (matched normal tissue)10 are considered. Using the two signature sets included with the deconstructSigs package (Top: the original Nature 2013 signatures2. Bottom: the COSMIC11 signatures) both methods identify the same signatures as being active and produce estimates of contribution weight that are remarkably similar. Note that we are not adjusting for frequencies of contexts in the genome in these analyses.\n\nThe disadvantage of the ILM approach comes from its having to define a subset of signatures to include in the model. While the signature matrix is of full rank, with noise in the system it is sometimes possible to approximate an observed vector with several different linear combinations of signatures, and an ILM approach is not guaranteed to give consideration to the correct combination of signatures. Even if the correct solution is reached, it can be a substantially slower approach. It is not difficult to simulate a combination of signatures that takes thousands of iterations and thousands of times longer to run than the QP approach.\n\nIf one simulates a flat combination of all available signatures, then the ILM approach performs worse than the QP approach. A fairer comparison would be to consider all equal combinations of just two signatures (with noise added). Of 351 possible such combinations using the Nature 2013 signature set2,5, the majority are well inferred using both the ILM and QP approaches, while one (the combination of signatures 1B and 3) performs poorly for both methods. Aside from these, there is a definite set of combinations for which the ILM approach performs markedly worse than the QP approach (See Figure 2). Pairs involving signature 1B, or signature 5, appear to cause the most problems. It is not the case that the problematic pairs are themselves highly correlated, but the 1B and U2 signatures are, possibly explaining the outlying nature of the U2-R2 pair. This exercise took approximately 5 seconds using the QP approach, and approximately 15 minutes using the ILM approach (on a well-specified desktop).\n\nA. 351 simulated datasets were constructed, one for each possible pair of the 27 Nature 2003 signatures, with equal weighting given to both of the signatures and independent uniform errors applied to each mutational context count (ranging from –5% to +5%). The contributions for the two signatures that should be detected are illustrated, with a line linking the estimates from the ILM and QP methods. Perfect performance would see contributions of 0.5 estimated for both signatures in all cases. The identities of outlying signature-pairs are indicated. B. The contributions estimated from the combination of signatures 13 and 5. C. The contributions estimated from the combination of signatures 2 and 5. D. The contributions estimated from the combination of signatures 1B and R2. E. The contributions estimated from the combination of signatures R2 and U2. In all four cases, both methods underestimate the contribution of one signature, but the ILM method more drastically. The ILM method is also more prone to the erroneous detection of other signatures.\n\n\nConclusion\n\nSince it makes use of well-established and core R code in a classical mathematical context, no new software is required to use the QP approach (see Data and software availability and Supplementary material for details of implementation). The speed and improved performance of the QP approach makes it an attractive alternative to the ILM method and complements the additional functionality of the deconstructSigs package5.\n\n\nData and Software Availability\n\nF1000Research. Dataset 1: An R Markdown document that when compiled will reproduce all the results presented, 10.5256/f1000research.8918.d12418113.\n\nThe raw oesophageal adenocarcinoma data for library SS6003314, from which some of these counts are derived, are available from the European Genome-phenome Archive (EGA; accession EGAD00001000704).",
"appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nAGL was supported in this work by a Cancer Research UK programme grant [C14303/A20406] to Simon Tavaré.\n\nAGL acknowledges the support of the University of Cambridge, Cancer Research UK and Hutchison Whampoa Limited. Whole-genome sequencing of oesophageal adenocarcinoma was part of the oesophageal International Cancer Genome Consortium (ICGC) project. The oesophageal ICGC project was funded through a programme and infrastructure grant to Rebecca Fitzgerald as part of the OCCAMS collaboration.\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\nWGS sequencing pipelines for the oesophageal ICGC project are managed by Lawrence Bower.\n\n\nSupplementary material\n\nThe R Markdown document (Dataset 1) compiled into a PDF file.\n\nClick here to access the data.\n\n\nReferences\n\nPfeifer GP, Denissenko MF, Olivier M, et al.: Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers. Oncogene. 2002; 21(48): 7435–7451. PubMed Abstract | Publisher Full Text\n\nAlexandrov LB, Nik-Zainal S, Wedge DC, et al.: Signatures of mutational processes in human cancer. Nature. 2013; 500(7463): 415–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShiraishi Y, Tremmel G, Miyano S, et al.: A Simple Model-Based Approach to Inferring and Visualizing Cancer Mutation Signatures. PLoS Genet. 2015; 11(12): e1005657. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGehring JS, Fischer B, Lawrence M, et al.: SomaticSignatures: Inferring mutational signatures from single-nucleotide variants. Bioinformatics. 2015; 31(22): 3673–3675. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRosenthal R, McGranahan N, Herrero J, et al.: DeconstructSigs: delineating mutational processes in single tumors distinguishes DNA repair deficiencies and patterns of carcinoma evolution. Genome Biol. 2016; 17(1): 31. 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. Reference Source\n\nS original by Berwin A. Turlach R port by Andreas Weingessel: quadprog: Functions to solve Quadratic Programming Problems. R package version 1.5-5. 2013. Reference Source\n\nGoldfarb D, Idnani A: Dual and Primal-Dual Methods for Solving Strictly Convex Quadratic Programs. Lect Notes Math. Springer-Verlag, 1982; 909(i): 226–239. Publisher Full Text\n\nGoldfarb D, Idnani A: A numerically stable dual method for solving strictly convex quadratic programs. Math Program. 1983; 27(1): 1–33. Publisher Full Text\n\nWeaver JM, Ross-Innes CS, Shannon N, et al.: Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis. Nat Genet. 2014; 46(8): 837–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBamford S, Dawson E, Forbes S, et al.: The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website. Br J Cancer. 2004; 91(2): 355–358. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPutnam NH, O'Connell BL, Stites JC, et al.: Chromosome-scale shotgun assembly using an in vitro method for long-range linkage. arXiv: 1502 . 05331v1 [ q-bio . GN ] 18 Feb 2015. Genome Res. 2016; 26(3): 342–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLynch A: Dataset 1 in: Decomposition of mutational context signatures using quadratic programming methods. F1000Research. 2016. Data Source"
}
|
[
{
"id": "16433",
"date": "06 Oct 2016",
"name": "Mohamed Helmy",
"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 by Lynch presents a technical improvement of a recently published method1 for inferring signatures of mutational contexts from large cancer sequencing data sets. The author proposes a quadratic programming (QP) approach over the iterative linear modeling (ILM) approach that was implemented in Rosenthal et al. According to the article, the presented approach provides technical improvement (speed) as well as an improvement in the accuracy.\n\nThe paper is well written and the results support the technical improvement of the QP approach over the ILM approach. The exercise provided by the author shows ~180 folds increase in the speed when using QP, comparing with the ILM approach (5 seconds vs. 15 minutes, respectively). That is a significant increase in performance that can be very useful when using such data in clinical applications, for instance. However, the current manuscript is not showing an improved accuracy for the QP approach.\n\nTherefore, I would recommend adding more details that show the improvement in accuracy or just focus only on the improved performance of the presented approach.\n\nAlso, I have one minor comment: for consistency, Figure 1 should be A and B instead of top and bottom.",
"responses": []
},
{
"id": "17440",
"date": "17 Nov 2016",
"name": "Miguel Vazquez",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 tackles the problem of determining the mutational processes that were active on a tumor, and specifically in a single sample setting by leveraging already available signatures. The relevance of this approach is thus clear and was established in previous work: it allows working with signatures in a more general setting such as the clinic, and reusing already available signatures helps interpretation by the community as these become more familiar to all.\n\nThe author's contribution is limited to a technological advance, but in that is seems to surpass the previous approach in speed and accuracy (though I present some reservations below) by casting the problem into the more sophisticated framework of quadratic programming. I think this approach has benefits and I'm convinced that at no expense, and as such, I'm strongly favorable. I have however a few concerns that I'd like to raise.\nBiologically I understand that mutational processes have signatures that are non-orthogonal, so a particular footprint of activity (the mutations on a sample) could in general be explained by different activation patterns of these signatures. How do these methods account for prior probabilities? e.g. mutational patterns related to smoking can be far more prevalent that exposure to a rare carcinogenic that could resemble the smoking signature in whole or in part. I can imagine the methods that extract this signature leveraging cohort data to untangle these prior probabilities, but then I think the methods presented in this paper in the deconstructSig cannot make use of this priors. In any case, I don't think current cohort methods predicting de-novo signatures are accounting for these priors since I would imagine they should be reporting these in addition to the signatures, which I believe they are not.\nComing back to the article at hand, the second paragraph in the result section seems to relate to this question in part. I find this paragraph confusing, possibly due to my own shortcomings so perhaps the author can clarify it for me, or even make it more clear on the text if need be. Let me explain. The author claims that the signature matrix is full rank. Correct me if I'm wrong, but in general it need not be, making the problem of approximating the result with different combinations is not just a result of noise and actually not specific to ILM, but to both methods. In fact the following phrase: 'an ILM approach is not guaranteed to give consideration to the correct combination of signatures' seems unfair, does the QP approach offer such guarantees? If so, perhaps this could be explained.\nThis paragraph was one of the main arguments for the improvement on accuracy, and I've presented my reservations. The other argument are some experiments presented on synthetic data involving the mixture of two signatures. These experiments seem too simplistic and I believe they do not address the problems presented in the previous paragraph either. However I do find that they suffice for the purposes of this article.\nIn conclusion, I concur with Mohamed that its mostly the performance that drives the message home at this point. Though I would not like to discourage indexing of this article, I feel that the author could improve his arguments regarding accuracy.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1253
|
https://f1000research.com/articles/5-61/v1
|
13 Jan 16
|
{
"type": "Observation Article",
"title": "Bringing experiential learning into the lecture theatre using 3D printed objects",
"authors": [
"David P. Smith"
],
"abstract": "The ability to conceptualize 3D shapes is central to understanding biological processes. The concept that the structure of a biological molecule leads to function is a core principle of the biochemical field. Conceptual teaching usually involves vocal explanations or using two dimensional slides or video presentations. A deeper understanding may be obtained by the handling of objects. 3D printed biological molecules can be used as active learning tools to stimulate engagement in large group lectures. These models can be used to build upon initial core knowledge which can be delivered in either a flipped form or a more didatic manner. Within the teaching session the students are able to learn by handling, rotating and viewing the objects to gain an appreciation of an enzyme’s active site or the structure of DNA for example. Models and other artefacts are handled in small groups and act as a focus for talking points to generate conversation. Through this approach core knowledge is first established and then supplemented with high level problem solving through a \"Think-Pair-Share\" cooperative learning strategy. The teaching delivery is adjusted based around experiential learning activities by moving the object from mental cognition and into a physical environment leading to student engagement in the lecture theatre and a dialog with the lecturer. The use of artefacts in teaching allows the lecturer to create interactive sessions that challenge and enable the student. This approach can be applied at all levels and across many disciplines.",
"keywords": [
"3D printing",
"Active Learning",
"Experiental Learning",
"Higher Education",
"Biomolecules"
],
"content": "Introduction\n\nAbility to conceptualize 3D shapes is central to the understanding of biological processes. The dogma that the structure of biological molecules leads to function is central to biochemical understanding and is a core principle of the field. For example how the binding site of enzymes catalyses a reaction or how the major groove of DNA allows specific interactions with transcription factors. Understanding of such concepts is often a requirement for accreditation by learned bodies such as the Society of Biology, 2013 and the Institute of Biomedical Science, 2010 (https://www.ibms.org/go/qualifications/ibms-courses/accreditation). Grounding in these concepts is often undertaken during the first year of study on the undergraduate courses within core modules in large group teaching environments. Students arrive with a range of experiences and prior knowledge ranging from the basic to more in-depth understanding of these topics.\n\nEysenck (2012) explains that within the teaching space students are required to imagine what would happen if an object was rotated or altered in a process known as \"mental rotation\". Although some students have the ability to picture 3D objects in their minds, this is not true for all. Even at higher levels fellow researchers have stated that they \"can't conceptualise protein shapes in my mind\".\n\nTraditionally the knowledge required to understand 3D structure and related concepts have been presented through the use of PowerPoint slides; slides which are often heavy in text. PowerPoint slides represent objects two dimensionally and this is useful for detailing core knowledge. This approach however does not help the students develop more complex cognitive 3D mental rotation skills (Nigel, 2014). There is a danger that the activities are perceived to be content driven by using a two dimensional approach in which the students remain passive observers throughout the session. This approach affords a more superficial engagement with the subject, potentially promoting ‘surface learning’ behaviour, leading to poorer understanding (Biggs, 1999). This behaviourist approach has its merits under certain conditions such as when a large amount of content needs to be covered in a short amount of time (Woolfolk, 2009). However, this approach is restricted to the acquisition and learning of knowledge and can prevent access to higher tiers of learning (Anderson & Krathwohl, 2000; Bloom, 1956). An alternative to this approach is the inclusion of active learning in teaching sessions whereby students become involved in the learning and are engaged in activities leading to higher order thinking (analysis, synthesis, evaluation) (Bonwell, 1991). Presented here is one such approach where students handle physical 3D printed objects within a teaching setting.\n\n\nMethodology\n\nWithin the research setting physical structural models of molecules have long-been used to help understand function. Models of the protein in question are often generated and handled in small group meetings as talking points to generate new hypotheses. This approach was adapted to large group teaching sessions with cohorts of 150 students in a standard tiered lecture theatre. Sessions using the models were delivered twice to the same students on their first year of study. Once in their first semester and once in the second semester of a two semester core Biochemistry module. The models have also been used in second year teaching when discussing DNA binding proteins within a Molecular Biology module. To encourage students to become engaged in the learning existing sessions were adapted to deliver base level learning supplemented with high level problem solving through the use of 3D printed models. The 3D printed molecules in question were linked to the core content and act as a focal point for learning. Models where created from the protein data bank (PDB) code 2LYZ (Lysozyme) and B-form of DNA was taken from the now defunct Glactone Pedagogical PDB collection. The PDB file was modified by the removal of the water molecules and the surface of the molecule was calculated in a molecular graphics program (Visual Molecular Dynamics 1.8.5). PDB files are also included as Supplementary material 1 and Supplementary material 2. The resulting files where rendered in a standard STL format using the (STL Plugin, Version 2.0) which is compatible with CAD and most 3D printers. STL files are also included as Supplementary material 3 and Supplementary material 4. Models used here were produced on a fused deposition modeling (FDM) Dimension sst 768 rapid prototyping 3D printer (Figure 1) and were approximately 2 × 2 × 4 cm. Paper based stereo images were also provided in the same session. The use of the 3D projection images also allowed the students to review and reflect on the learning at a later date and gave a focal point and prompt for later revision (Figure 2).\n\nB-form DNA (right) and the enzyme lysozyme PDB: 2LYZ (left) used within the teaching session.\n\nCross eye stereo image: Instruction to students were gaze at the stereo pair, keeping your eyes level (don’t tilt your head left or right), and cross your eyes slightly so that the two images in the center come together. When they converge or fuse, you will see them as a single 3D image.\n\nSessions were structured so that taught content prepared the students for the experiential learning activities by first establishing core knowledge. This content gave the students the vocabulary to later describe the objects they would handle. The taught content laid foundation knowledge relating to how molecules such as enzymes perform reactions and to gain an appreciation of the structure of DNA. The active learning component was then included to placing the object out from mental cognition and into a physical environment. This was achieved by allowing the students to rotate and view objects physically through the handling of 3D printed models of these biomolecules. The overall teaching style follows a simplified form of Kolb’s experiential learning cycle (Kolb, 1984). This model is well-established in science based learning. As teachers and learners we are able to jump onto the cycle at any point but in order for it to be useful the stages must be followed in sequence. Learning can then be applied in new situations and subsequently built upon.\n\nNew concepts were introduced through the use of slides, videos and written material. A range of media animations, web-based content and strong links to core texts were used. The \"thinking\" section of the lesson plan had prepared the students to identify key features of the models they would later handle.\n\nIn order to develop a 3D understanding of biomolecules students were asked to handle printed models and apply their new knowledge and concepts through self-directed small group discussions (Figure 3). Questioning was centred on those features they could observe and was objective, such as: What do they feel like? What general shape do they have? What features can you observe? This encouraged student interaction as there was no wrong answers to the questions as it was personal observation.\n\nStudents handle the 3D printed molecules and were asked to identify structural features.\n\nThrough this approach core knowledge is first established which is then supplemented with high level problem solving through \"Think-Pair-Share\" cooperative learning strategies. Students are asked to think through questioning about an aspect of the object and discuss the answers with each other. Questions were asked that probed understanding, such as what are those bumps on the surface? What is the function of that groove? As such, learning is enhanced through the opportunities to elaborate on the ideas through conversation. I observed that this approach led to increased student engagement in the lecture theatre as the students are willing to talk with each other and the lecturer as confidence in their understanding increased.\n\nFinally the students are given time and encouraged to write on handouts in their own words the key points and note theories that have been discussed. (Figure 4). The handouts were structured such that the key learning objectives were recorded (handouts used can be found in Supplementary material 5). For example, students were asked to identify key features of the molecule in question and complete a question sheet where they were asked to identify structural features. In order for the students to take ownership of the knowledge, they discussed specific situations for how this information is used in practice. Examples where given from a research-informed context and were tailored to be course specific.\n\nHandouts where designed that allowed the students to identify key features of the molecule in question and complete a question sheet. This ensured key learning objectives were recorded.\n\n\nConclusion\n\nAccess to 3D printing technology is becoming more wide spread as the costs associated with the technology drop and most institutes already have access to such printers. Active learning approaches are becoming increasing common place as teaching staff move away from didactic strategies (DesLauriers et al., 2011; Seery, 2015; Sharples et al., 2014).\n\nThe use of objects within the classroom is one such approach and, in evaluations conducted with my own students, they identify them as both engaging and informative. Students describe helpful visual aids such as “scale models\" and \"engaging lectures\" with reference to previously produced 3D printed models. The students engage with the models which stimulate conversation rather than distract attention.\n\nThe use of objects, therefore, can be seen as a focal point for conversation and suggests there are similar applications to enhance other areas of teaching. Peers within the nursing team at my own university have considered the use of dolls as talking points for their students to support discussions about empathy. Such abstract learning environments dealing with relationships rather than facts and thinking situations in symbolic form can be pictured as an area of conceptual knowledge (Anderson & Krathwohl, 2000). Objects have also been used by peers in analytical chemistry teaching where parts of instruments help to develop understanding into drug detection when taken to the lecture theatre.\n\nThe use of artefacts in teaching opens new ways to challenge students. Teachers can create interactive sessions that challenge students to see artefacts through the lenses of mathematics, science, language, arts, and social studies. While the use of objects in both large and small group teaching is currently under researched and under reported, it has the potential to increase student engagement by facilitating active learning methods.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by the Department of Bioscience and Chemistry, Sheffield Hallam University, Sheffield, S1 1WB.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary material 1: Lysozyme PDB file. The file \"2LYZ no water.pdb\" contains the atomic coordinates of the enzyme Lysozyme with the crystallographic water molecules removed from the file. The file originated was sourced from the PDB file 2LYZ.\n\nClick here to access the data.\n\nSupplementary material 2: B-form DNA PDB file. The file \"bdna.pdb\" contains the atomic coordinates of B-form DNA used to create the DNA model structure and hand-out images. The file originated from the now defunct Glactone Pedagogical PDB collection.\n\nClick here to access the data.\n\nSupplementary material 3: Lysozyme STL file. The file \"lysozyme.stl\" contain surface renderings of lysozyme and was used to create the 3D printed model shown in Figure 1. The files is in a standard STL (STereoLithography) format native to the stereolithography CAD software created by 3D Systems. This file format is supported by many software packages and is widely used for rapid prototyping and 3D printing.\n\nClick here to access the data.\n\nSupplementary material 4: B-form DNA STL file. \"B-DNA2.stl\" contain surface renderings of B-form DNA and was used to create the 3D printed model shown in Figure 1. The files is in a standard STL (STereoLithography) format native to the stereolithography CAD software created by 3D Systems. This file format is supported by many software packages and is widely used for rapid prototyping and 3D printing.\n\nClick here to access the data.\n\nSupplementary material 5: Hand-outs used to allow students to record their experiences and notes from the session.\n\nClick here to access the data.\n\n\nReferences\n\nAnderson LW, Krathwohl DR: A Taxonomy for Learning, Teaching, and Assessing: A Revision of Bloom's Taxonomy of Educational Objectives. Allyn & Bacon. 2000.\n\nBiggs J: Teaching for quality learning at university. Buckingham: Society for Research into Higher Education and Open University Press. 1999.\n\nBloom BS (ed.): Taxonomy of Educational Objectives: the classification of educational goals. Handbook I: Cognitive Domain. New York: McKay. 1956. Reference Source\n\nBonwell CC, Eison JA: Active learning: Creating excitement in the classroom. ASHE-ERIC Higher Education Report No. 1. Washington DC: The George Washington University. 1991. Reference Source\n\nDeslauriers L, Schelew E, Wieman C: Improved learning in a large-enrollment physics class. Science. 2011; 332(6031): 862–864. PubMed Abstract | Publisher Full Text\n\nEysenck MW: Fundamentals of cognition. Psychology, Hove. 2012. Reference Source\n\nInstitute of Biomedical Science: Criteria and Requirements for the Accreditation and Re-accreditation of BSc (Hons) degrees in Biomedical Science. 2010; [Accessed: 28th July 2014]. Reference Source\n\nKolb D: Experiential learning: experience as the source of learning and development. Englewood Cliffs, New Jersey: Prentice Hall. 1984. Reference Source\n\nNigel JT: \"Mental Imagery\". The Stanford Encyclopedia of Philosophy. Edward N. Zalta (ed.). 2014. Reference Source\n\nSeery MK: Flipped learning in higher education chemistry: emerging trends and potential directions. Chem Educ Res Pract. 2015; 16: 758–768. Publisher Full Text\n\nSharples M, Adams A, Ferguson R, et al.: Innovating Pedagogy 2014: Open University Innovation Report 3. Milton Keynes: The Open University. 2014. Reference Source\n\nSociety of Biology: Handbook for the Society of Biology’s Degree Accreditation Programme. 2013; [Accessed: 28th July 2014]. Reference Source\n\nWoolfolk A: Educational Psychology: Eleventh Edition. Prentice Hall. 2009. Reference Source"
}
|
[
{
"id": "11937",
"date": "26 Jan 2016",
"name": "Richard Bowater",
"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 provides a brief description of an attempt to introduce experiential learning into lectures in the biochemical sciences. As described below, the author highlights a fundamental aspect of the subject that some students struggle to understand fully and proposes an approach that aims to help students engage better with the topic and the issues involved. Structures of biological molecules are critical to their function and this is a core principle that must be understood by all students of the field of biochemistry. Full appreciation of this link requires students to develop 3-dimensional knowledge of a molecule and relate this to its biochemical activities. These are challenging concepts to introduce to students, especially in large cohorts. This article describes a strategy that takes advantage of the increasing availability and cost effectiveness of 3D printers to prepare molecules that can be “handled” by the students. The 3D printed biological molecules are used as active learning tools to stimulate student engagement and a deeper understanding of the link between structure and biochemical function. The approach described in the article is that core knowledge should first be established through conventional teaching approaches. The teaching delivery is then supplemented by discussions among the students and lecturer about the printed molecules, allowing interrogation of its physical environment. The article provides an appropriate discussion of a topic that is clearly challenging to inexperienced learners in the biochemical sciences and suggests an approach that goes beyond the conventional lecture. However, there is no evaluation of whether the approach is successful or cost-effective and, thus, readers cannot easily judge if they should consider it in their own teaching environment. There is a comment that the author “observed that this approach led to increased student engagement in the lecture theatre as the students are willing to talk with each other and the lecturer as confidence in their understanding increased”, but there is no direct feedback from students involved in the sessions. The article suggests that its approach would be of value to other disciplines. In fact, the use of artefacts and the potential to handle objects is frequently used within the arts and humanities, particularly in subjects that combine history and art. The benefits of such approaches have been well described in the pedagogical literature for these disciplines and this article would be improved if it provided a deeper review and analysis of such information. To be of improved value to the biochemistry teaching community, this study needs to provide a much clearer hypothesis. What is (are) its fundamental aim(s) and how will its success be tested and evaluated? It would be beneficial if a study could be developed that tested whether inclusion of the printed molecules in teaching sessions led to improved biochemical understanding among the students involved. Clearly this could take a significant amount of time and effort and may be beyond the remit of the study that is described. Whether or not it is possible to evaluate the success of the approach, there are a number of important details that would be useful to assist other educators in considering whether they should adopt the strategy. For example, how many molecules are needed for each group of student and what is the approximate cost of preparing the molecules? Is there any value in using the approach multiple times (with different molecules) within the same module or same cohort of students? In relation to publication of this article, it is not clear that it has been submitted as the most appropriate type of article for F1000Research. The study has been submitted as an Observation Article and the guidelines to authors state that: “Observation Articles allow the description of a novel observation that may be unexpected, and possibly currently without explanation.” It is not clear to me what details in the article are “unexpected” or “without explanation” - these points need to be made much clearer to the reader. In considering the different types of articles published within F1000Research, it seems that it may be more applicable as a Research Note. The article is reasonably clear and well written, but there are some minor errors that should be corrected by careful proof-reading. The title could be improved by highlighting that it focuses on “biochemistry teaching”. In summary, this article describes an interesting but rather straight forward intervention to allow biochemistry students to gain a better appreciation of the link between molecular structure and function. The suggested approach makes good use of computer-based technologies that are becoming more accessible and it is thus likely to be of interest to a wide group of educators in the biosciences (and other disciplines). Unfortunately, the study does not present any evaluation of the effect of the approach on student learning or understanding. Such information should be obtained from further cohorts of students in order to enable other educators to consider the value in the approach.",
"responses": [
{
"c_id": "1981",
"date": "03 Jun 2016",
"name": "David Smith",
"role": "Author Response",
"response": "Thank you for your comments and by addressing them I hope the manuscript has been improved. In the response below I have set out the changes made.The work is more than using 3D printed objects, it is student-centred education, interactive and motivating and specially thought to address a problem in science, to enable visualization of abstract concepts, specifically in Biochemistry.Thank you for your suggestions and I have altered the title to reflect your comments. \"Active learning in the lecture theatre using 3D printed objects to visualise abstract concepts.\"Many teachers also use 3D visualization programs, combining theoretical and computer laboratory sessions. Differences, advantages and disadvantages between these two approaches could be discussed.The use of computer based representations is now easily accessible via personal devices. There are a wide range of 3D visualisation programs available which enable molecular structures to be moved, altered, rotated and interrogated. Programs such as the Java based Jmol applet allow students to manipulate molecules and investigate their structures. There are also a range of free programs that allow students to visualise molecules on smart phones and tablets such as \"Molecules\" for Apple iOS and \"ESmol\" for Android devices. These programs offer many advantages such as ready access to the > 118500 structures available in the protein data bank (PDB). However, their use as tool in large group teaching can be problematic as all students require access to a device. In addition providing technical support to 150 students across three different platforms within a lecture theatre can also be problematic. Molecular viewers do work very well in small group seminar or lab sessions where support can be given or devices can be provided. Students can be directed through a range of tasks and the molecules can be explored in greater depth (Harris, 2009). However, the tablet based applications lack the tactile aspect of physically handling the object in question and can prevent the abstract observations that generate the initial interactions. The models are also engaging and can be used by groups of people who have had little training, unlike the visualisation programs (Harris, 2009). The models also allow an ability to quickly make abstract observations for example the spiral nature of DNA or the large clefts on an enzyme. It is these abstract observations that stimulate the initial peer-to-peer conversations. This approach allows the students to remain engaged with the topic and revisit their observations after the teaching session. In a study in which 3D printed models were offered alongside a molecular imaging program the students tend to prefer the models when asked questions about molecular structure that required higher order thinking skills (Harris, 2008). The tactile nature of the models appears therefore to leads to a more lasting memory of the interaction (Hurman, 2006). The response to the following questions has been embedded directly into the text.What was actually done? Molecules were handed around the group by the lecturer. They were handed out starting at the end of each of the rows of students. The students were asked to pass the molecules around and handle them directly. Once most of the students had handled the objects they were encouraged to talk with their peers about their observations.Why were those structures chosen? The two structures were chosen as they these molecules are established as examples within the taught material on the course. DNA is central to molecular biology and an understanding of how other molecules such as transcription factors interact with it is a key learning point in the Biochemistry program. Lysozyme was chosen as its structure and function are well understood. It also has a defined active site in which the substrate has been modelled. The use of lysozyme also integrated well with the rest of the module. Its action on the lipid polysaccharides found on the cell wall of gram negative bacteria resulting in lysis allowed links to microbiology and protein purification. It also integrates well with a second year structural biology practical in which lysozyme can be induced to undergo change from an alpha / beta structure to the beta sheet rich structure of amyloid fibrils resulting in a loss of function.Which were the concepts students were expected to learn in each session? The main concepts that the students were expected to learn was that the structure of a molecule brings about its function and subsequent properties.What were the related learning outcomes? In the DNA sessions the main learning outcome was to understand the difference between the major and minor grove within the structure of DNA and how proteins interact with these groves. Within the lysozyme sessions the learning outcome was to be able to identify where an active site maybe located on a protein and how a substrate would interact with it.How many groups? The session has been run twice a year for three consecutive years with ~120 - 150 students being present at each session.How many molecules? Within each session 20 molecules were used.Was the discussion tutored? If it was, how many tutors participated? The sessions were run in a standard lecture theatre with one lecturer delivering the session.The models seem very small, what were the structural features they had to identify mentioned in figure 3? For the DNA molecule it was the major and minor grove. The models are scaled such that an index finger would fit into the major grove. In the case if the enzyme it was the active site located across the middle of the protein.Costs?£7 each for raw materials As this experience was justified as a mean for conceptualizing 3D shapes for understanding Biochemistry, other reported experiences using molecules should be discussed and compared.The following text has been addedThe use of physical models as a learning tool have been used to depict molecular geometry in both biochemistry and chemistry (Dori, 2001). The benefit of these objects has been demonstrated in an analysis of visuospatial thinking in chemistry. In this study it was concluded that adept visual perception skills correlate with achievement. DNA and protein models assembled from coloured computer-printouts on transparency film sheets have also been demonstrated to aid students in grasping various aspects of biopolymers (Jittivadhna, 2010). Students who hold physical models in their hands gain a better understanding of molecular geometry, than they could achieve solely from viewing images on a printed page. It has been reported by both Herman (2006) and Bain (2006) that physical models that can be easily manipulated, can play an important role in capturing the interest of students and encouraging deeper sophisticated thinking (Bain, 2006). Additionally, the students gain a language for talking about the concepts in question and enhance their understanding of abstract concepts by handling models (Wu, 2004).The positive comments from students polled in this study, in which 3D printed models were rated as most useful, are echoed in a comparable study where a side-by-side comparisons of seven different learning tools were undertaken by students in an introductory biochemistry class (Roberts, 2005). In that study, the models were shown to have an important role in capturing the interest of the students and stimulated sophisticated questioning. The main difference between these observations and those presented here, relate to the learning environment. Within the side-by-side comparisons study, the core concepts were introduced in a lecture and the models were handled as part of a 3-wk laboratory in a group of twenty students, generating tangible learning gains were observed. In the study discussed here, however, 150 students were allowed to handle the models directly within the lecture environment. Handling the models aided the students in visualising the molecules demonstrating the applicability of this approach to large group teaching. Schönborn, (2006) states that the ability to visualise ideas is a key skill for all students and it is a key skill for biochemists who are often presented with a range of visual interpretation including drawings, images, dynamic visuals, animated visuals, multimedia, and virtual reality environments. The use of models has the potential to help students construct their own visualisation and understanding of these molecules as demonstrated by the student comments reported here.The main weakness of this work is that no assessment of the approach is included. On the advice of both reviewers the perception of the students was assessed via a written questionnaire. Students in their final year of study were asked to reflect on the use of the models during their past learning. 44 students were questioned and 35 remembered using the models. 32 of these responded with positive comments and 3 with neutral comments. Of the 9 students who did not remember using the models many admitted to not having attended the lecture. The following text and supplementary information has now been added. In addition comments have been referred to in the discussion section. ResultsStudents in their final year of study were asked to reflect on the use of the 3D printed models during their past learning. 44 students were asked \"Did you find the models helpful, if so how?\" through an anonymous survey. Of the 44 responses 35 remembered using the models and 32 of these responded with positive comments (3 with neutral comments). Of the 9 students who did not remember using the models many admitted to not having attended the lecture.Some students commented on the benefits the models provided as a visual aid,\"they were very useful for highlighting the key lecture points as well as being a visual aid.\"Student comments also highlighted the use of the models as a tool in understanding the key learning objectives,\"Very useful to help understand major and minor grooves\", and \"Allowed us to visualise the major and minor groove of DNA, as well as the binding sites for enzyme\". The students also explained that the models provided an alternative way of presenting information,\"they gave a 3D better understanding of the 3D structure of the enzyme than a 2D computer image.\"It was also noted that the models could be used to gain a sense of scale, for example,s the difference in size between a ribosome and organelles. The full list of comments is included as supplementary information. During each of the sessions there was a high level of student engagement involving the students talking to each other and to the lecturer with the key learning points being recalled three years after the teaching session.Did the students seem motivated? Yes there was a high level of engagement during the sessions; the students were talking to each other and to the lecturer.Did they participate more actively than in other activities? The participation level was probably equivalent to other sessions. However I have noted that a range of activities within the sessions a good way to keep students motivated and engaged. Too much of any intervention can lead to disengagement as has been reported for flipped classroom approaches.Did they comment informally positive aspects?See above responseAnother approach could be to analyze exams answers regarding the concepts that were studied in these special sessions and compared them to those of the previous year.Unfortunately we do not hold the exam scripts locally and so gaining access to this is not possible. However it will be built into further studies as it is a good suggestion."
}
]
},
{
"id": "12885",
"date": "14 Mar 2016",
"name": "Carola Bruna",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents a pedagogical experience in which 3D printed objects were used to promote the understanding of biochemistry concepts in two student-centred interactive sessions within a course. The design is based in the advances and availability of 3D printing technology and the importance of conceptualizing three-dimensional structure in order to understand biochemistry core concepts such as the relationship between the structure and function of proteins. 3D printing technology is in fact becoming increasingly accessible and it is contributing and changing many fields. Undoubtedly, education should benefit from this technology, probably many teachers are thinking about it, including myself. Thus, experiences on how to implement it in class are interesting and valuable. In this sense, this work can contribute with relevant information. Nevertheless, some aspects need to be addressed, clarified or discussed further so that weaknesses and strengths are identified, which will allow replicating or improving the shared experience. In this context, the following suggestions could improve this manuscript: Title: The work is more than using 3D printed objects, it is student-centred education, interactive and motivating and specially thought to address a problem in science, to enable visualization of abstract concepts, specifically in Biochemistry. The author state that 3D structures and related problems have been presented with slides. Many teachers also use 3D visualization programs, combining theoretical and computer laboratory sessions. Differences, advantages and disadvantages between these two approaches could be discussed. The experience should be described further. What was actually done? Why were those structures chosen? Which were the concepts students were expected to learn in each session? What were the related learning outcomes? How many groups? How many molecules? Was the discussion tutored? If it was, how many tutors participated? The models seem very small, what were the structural features they had to identify mentioned in figure 3? Costs? As this experience was justified as a mean for conceptualizing 3D shapes for understanding Biochemistry, other reported experiences using molecules should be discussed and compared. For example: “The effect of student manipulation of molecular models on chemistry achievement according to Piagetian level” or “Using three-dimensional models to teach molecular structures in high school chemistry”. I am sure there are more examples, maybe even in biochemistry. The main weakness of this work is that no assessment of the approach is included. I understand that it is very difficult to assess learning gain, and that the intention of the author is modest, in the sense that this article is intended to share an experience instead of researching education. However, at least the perception of the students should have been included. This would at least suggest the strongest aspects and allowed identifying possible improvements. If the intervention is recent and there still contact with the students, there is still a possibility to conduct an online survey. If that is not the case, at least the author should comment further regarding the perception of the teacher: Did the students seem motivated? Did they participate more actively than in other activities? Did they comment informally positive aspects? Another approach could be to analyze exams answers regarding the concepts that were studied in these special sessions and compared them to those of the previous year. Despite the previous comments, I still consider that sharing this experience is valuable to the community. In this work, I particularly appreciate that the activity was implemented in a large course using a standard classroom, with no additional requirements. If the main suggestions could be addressed, I believe it would be appropriate for indexing as published in F1000Research.",
"responses": [
{
"c_id": "1980",
"date": "03 Jun 2016",
"name": "David Smith",
"role": "Author Response",
"response": "Thank you for your comments and by addressing them I hope the manuscript has been improved. In the response below I have set out the changes made. Some off the comments made are reflected by referee 2 and the response below is applicable to both referees. Comment - However, there is no evaluation of whether the approach is successful or cost-effective and, thus, readers cannot easily judge if they should consider it in their own teaching environment.The cost per model was around £7 when purchased internally which covered materials. The models have now been used over four years in two different streams (Biology and Chemistry). There 20 models were produced with a total cost of £140. This is less than the printing cost of the handouts for the same session. Comment - There is a comment that the author “observed that this approach led to increased student engagement in the lecture theatre as the students are willing to talk with each other and the lecturer as confidence in their understanding increased”, but there is no direct feedback from students involved in the sessions.On the advice of the reviewer the perception of the students was assessed via a written questionnaire. Students in their final year of study were asked to reflect on the use of the models during their past learning. 44 students were questioned and 35 remembered using the models. 32 of these responded with positive comments and 3 with neutral comments. Of the 9 students who did not remember using the models many admitted to not having attended the lecture. The following text and supplementary information has now been added. In addition comments have been referred to in the discussion section. ResultsStudents in their final year of study were asked to reflect on the use of the 3D printed models during their past learning. 44 students were asked \"Did you find the models helpful, if so how?\" through an anonymous survey. Of the 44 responses 35 remembered using the models and 32 of these responded with positive comments (3 with neutral comments). Of the 9 students who did not remember using the models many admitted to not having attended the lecture.Some students commented on the benefits the models provided as a visual aid,they were very useful for highlighting the key lecture points as well as being a visual aid.\"Student comments also highlighted the use of the models as a tool in understanding the key learning objectives,\"Very useful to help understand major and minor grooves\", and\"Allowed us to visualise the major and minor groove of DNA, as well as the binding sites for enzyme\".The students also explained that the models provided an alternative way of presenting information,\"they gave a 3D better understanding of the 3D structure of the enzyme than a 2D computer image.\" It was also noted that the models could be used to gain a sense of scale, for example, the difference in size between a ribosome and organelles. The full list of comments is included as supplementary information. During each of the sessions there was a high level of student engagement involving the students talking to each other and to the lecturer with the key learning points being recalled three years after the teaching session. Comment -The article suggests that its approach would be of value to other disciplines. In fact, the use of artefacts and the potential to handle objects is frequently used within the arts and humanities, particularly in subjects that combine history and art. The benefits of such approaches have been well described in the pedagogical literature for these disciplines and this article would be improved if it provided a deeper review and analysis of such information. The following text has been integrated into the introduction discussing object based learning theory. The handling of molecular models within teaching sessions can aid this mental rotation and the use of models falls within the theories of object based learning. This approach involves the active integration of objects into the learning environment (Chatterjee, 2015). Working with objects strengthens learning is the central proposition of object-based learning as, according to Romanek (2008), the sense of touch can lead to a more memorable learning experience. The use of museums artefacts in history, art and biology has been well explored and there are a number of parallels between the handling of objects within these disciplines. Object- based learning theory links student activity to meaning making by challenging the student to engage with and interrogate the object It represents a constructivist approach in which the students develop their knowledge and understanding though interaction (Chatterjee, 2015). While the teacher facilitates this learning, the students have to learn for themselves through their interaction with each other centred on the object (Hannan, 2013). This approach enables the student to explore processes and events related to the object and further link these observations to complex abstract ideas and concepts.Findings from my study support Chatterjee's (2015) conclusion that the students gain real knowledge by being actively involved in the experience of handling the objects. It has been highlighted that object-based learning should be both mentally and physically stimulating through some form of problem solving or experimentation (Coffield, 2004) and this was achieved in this study by challenging the students to find the active site of Lysozyme or identify the region of DNA to which proteins bind.While objects can be used to enhance learning, there are logistical and pedagogical barriers to implementing them. For example, Cain (2010) says the main barrier for the implementation in the biosciences within the lecture theatre is the change to the adoption of a more student-centred and open-ended activities. The use of models in teaching needs to expand the concepts as understood by students at a particular stage in their learning (Bent, 1984) There is a danger for example when discussing enzymes that concepts such as the lock and key model could be reinforced over an induced fit model. Such dangers should be at the forefront of the lectures mind when utilising these models."
}
]
}
] | 1
|
https://f1000research.com/articles/5-61
|
https://f1000research.com/articles/5-353/v1
|
15 Mar 16
|
{
"type": "Review",
"title": "“White Paper” meeting summary and catalyst for future inquiry: Complex mechanisms linking neurocognitive dysfunction to insulin resistance and other metabolic dysfunction",
"authors": [
"Luke E. Stoeckel",
"Zoe Arvanitakis",
"Sam Gandy",
"Dana Small",
"C. Ronald Kahn",
"Alvaro Pascual-Leone",
"Aaron Pawlyk",
"Robert Sherwin",
"Philip Smith",
"Zoe Arvanitakis",
"Sam Gandy",
"Dana Small",
"C. Ronald Kahn",
"Alvaro Pascual-Leone",
"Aaron Pawlyk",
"Robert Sherwin",
"Philip Smith"
],
"abstract": "Scientific evidence has established several links between metabolic and neurocognitive dysfunction, and epidemiologic evidence has revealed an increased risk of Alzheimer’s disease and vascular dementia in patients with diabetes. In July 2015, the National Institute of Diabetes, Digestive, and Kidney Diseases gathered experts from multiple clinical and scientific disciplines, in a workshop entitled “The Intersection of Metabolic and Neurocognitive Dysfunction”, to clarify the state-of-the-science on the mechanisms linking metabolic dysfunction, and insulin resistance and diabetes in particular, to neurocognitive impairment and dementia. This perspective is intended to serve as a summary of the opinions expressed at this meeting, which focused on identifying gaps and opportunities to advance research in this emerging area with important public health relevance.",
"keywords": [
"Diabetes",
"insulin resistance",
"obesity",
"cognition",
"cognitive impairment",
"Alzheimer’s disease",
"vascular dementia",
"mechanism"
],
"content": "Background\n\nEmerging data have established links between systemic metabolic dysfunction, such as diabetes, and neurocognitive impairment, including dementia. The current epidemic of dementia is driven, at least in part, by the concurrent epidemics of obesity, insulin resistance, diabetes, and metabolic syndrome. Early research sought to elucidate the cause(s) for the apparent role of metabolic dysfunction in the increased prevalence of neurocognitive dysfunction and dementia, tentatively attributed to vascular contributions to cognitive impairment and dementia (VCID), and Alzheimer’s disease (AD).\n\nMore recent research has revealed that the relationships linking metabolism and brain dysfunction are bidirectional. On the one hand, diabetes increases risk of dementia by about two-fold, while on the other hand, the development of neurocognitive disorders has been linked to an increased risk of metabolic disease (Biessels et al., 2014). While the relationships linking metabolism and brain dysfunction in humans are more complex and more difficult to study in isolation, mouse models of isolated cerebral amyloidosis and cerebral amyloid angiopathy have been discovered to have peripheral insulin resistance (Ruiz et al., 2016).\n\nGenetic, epigenetic, environmental, and other mechanisms (likely in combination) are being implicated as underpinning these relationships and, in some cases, with behavior acting as a disease modifier. Of particular interest, both peripheral insulin resistance (the central pathophysiologic feature of type 2 diabetes) and “brain insulin resistance” (a much-discussed but poorly characterized entity) have been suggested to play important roles in neurocognitive dysfunction and dementia. Insulin resistance and the associated metabolic dysregulation can be driven by high-fat (“Western”) diet, physical inactivity, and obesity, and have deleterious effects on neurocognition (Ishii & Iadecola, 2015a).\n\nBecause the pathophysiological processes that lead to metabolic and neurocognitive dysfunction precede the development of clinical syndromes by years, early intervention and preventive measures are possible and an area of intense study (Norton et al., 2014). Indeed, AD neuropathology is detectable more than 10 years before the onset of the first clinical symptoms (Bateman et al., 2012) and pre-diabetes also precedes diabetes by years in most cases. There is now a focus on biomarkers and intervention research at pre-disease states, as there may be a higher chance that diseases are modifiable at this stage and prevention may be attainable (Sperling et al., 2015).\n\nConfounding the picture is evidence that diabetes worsens VCID and, in turn, that VCID increase the likelihood of developing AD pathology. Nonetheless, diabetes and dementia are complex diseases, with heterogeneous risk factors, underlying mechanisms, and clinical expressions, often with different contributing co-morbidities.\n\nIn this era of precision medicine, it is imperative that we identify the various metabolic-neurocognitive phenotypes in order to understand the mechanisms that drive these diseases so that we can develop targeted therapeutic strategies to successfully manage and, hopefully, prevent these complex, multifactorial diseases.\n\nIn July 2015, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health (NIH) gathered a group of cross-disciplinary scientific experts in Bethesda, MD for purposes of:\n\n(1) Reviewing the state-of-the-science;\n\n(2) Identifying key knowledge gaps and critical unanswered questions; and\n\n(3) Developing ideas for future directions to advance investigation at the intersection of metabolic and neurocognitive dysfunction.\n\nThe meeting focused on clarifying leading candidate mechanisms that may explain relationships between metabolic and neurocognitive dysfunction, with a special emphasis on the bidirectional relationships between diabetes and/or insulin resistance (either peripheral, central, or both) and cognitive impairment and AD and/or VCID. Although there has been increased attention to the neurodegenerative and/or vascular contributions to cognitive impairment and dementia (Snyder et al., 2015), it is important to consider the contributions of mechanisms that may not fall into either category as well as mechanisms that span multiple categories.\n\nIn this White Paper, we have attempted to survey and summarize the opinions among the experts in attendance with regard to the most likely mechanisms to investigate in order to improve our understanding of diseases and disorders at the interface of metabolism and cognition; in other words, to address the questions of “what do we know now?” and “what directions should be pursued in order to advance that knowledge?”. In so doing, we aim (a) to improve public health at the level of community medical care that considers patients within the context of their behaviors and environment; and (b) to inform public policy that would be required for a major effort toward prevention of these common co-morbid conditions of high societal burden.\n\n\nSummary of current knowledge\n\nThe high prevalence of type 2 diabetes (~26%) and dementia (~11%) in individuals > 65 years old, and the increased relative risk of all-cause dementia (~2 to ~2.5-fold) conferred by type 2 diabetes make understanding this connection a major public health imperative. Even individuals with type 2 diabetes without dementia have subtle impairment in performance (a third of a standard deviation, compared to individuals without diabetes) in a range of cognitive functions, including learning and memory, processing speed, and executive function across the lifespan (Biessels et al., 2014). These decrements in cognitive function are accompanied by structural and functional brain changes, with one estimate attributing five years of brain aging (based on volumetric magnetic resonance imaging, MRI) to diabetes relative to chronological age and estimates in those without diabetes (Franke et al., 2013). The most common neuroimaging findings include modest white and gray matter atrophy (most pronounced in the temporal and frontal cortices), cerebral small vessel disease, and disrupted structural and functional connectivity (Biessels & Reagan, 2015). Of relevance to understanding pathophysiologic mechanisms linking diabetes and neurocognition is that some of these brain changes may be observed prior to the development of diabetes (Brundel et al., 2014; Convit et al., 2003).\n\nThe neurocognitive profile of diabetes shares features with aspects of cerebrovascular disease and AD. However, it is still unclear if the relationship between diabetes and neurocognitive dysfunction and dementia is due to classic AD pathology, vascular processes such as infarcts or vessel pathology, other pathologies less closely related to AD or vascular disease (e.g., impaired brain insulin signaling), or some combination of these. Some have proposed that “all-cause” dementia be employed in order to avoid the implication that we can classify etiologies accurately based on current knowledge. Understanding the mechanism(s) that explain the neurocognitive complications of diabetes and other metabolic disease will be important if we are going to be successful in developing therapeutic targets and approaches to mitigate the effects of metabolic disease on the brain and cognitive function. This is especially the case if metabolic disease impacts the brain in ways that differ from its effects on other end organs impacted by diabetes and other metabolic disease.\n\nThe CNS pathology associated with metabolic disease has largely been characterized as a cognitive disorder with most of the research focused on brain regions and networks that support higher-order cognitive functions. Research on the central regulation of energy balance and peripheral metabolism, on the other hand, has almost exclusively focused on the hypothalamus and brainstem, brain regions and networks whose contributions to higher-order cognitive functions may not have been fully defined. The recent appreciation of the bidirectional relationship between diabetes and neurocognitive dysfunction/dementia presents an opportunity to combine efforts and resources to tackle both of these complex and related problems together. Cerebral amyloidosis can precede clinical cognitive decline for up to 30 years, and during this early phase may occur in limited regions of the brain. Therefore, it is plausible that AD pathology (amyloid-beta or Aβ, tauopathy) may be present in the hypothalamus prior to the onset of cognitive symptoms, and that this hypothalamic pathology could disrupt homeostatic functions such as energy balance and peripheral metabolism prior to the onset of detectable cognitive dysfunction (Ishii & Iadecola, 2015b).\n\nWhile diabetes is known to increase risk for dementia, the underlying mechanisms linking these conditions is less clear. Undoubtedly, given the well-established micro- and macro-vascular complications of diabetes, the least disputed mechanism of brain injury involves cerebrovascular disease. Indeed, it is now well-established that diabetes, and elevation in the most frequently used biomarker of diabetes (hemoglobin A1c), increases the risk of stroke, and that stroke in turn, increases the risk of cognitive impairment and dementia, including vascular dementia (Abbott et al., 1987; Banerjee et al., 2012; Gorelick et al., 2011; Ivan et al., 2004; Li et al., 2012; Ramirez et al., 2015). Aside from brain infarction, the central pathologic feature of stroke (Arvanitakis et al., 2006a), other vascular and related processes in the brain plausibly play a pathophysiologic role in relating diabetes to dementia, including white matter disease, breakdown of the blood brain barrier, inflammation, and others (Hsu et al., 2012; Shimizu et al., 2013). Also, co-morbid conditions to diabetes, such as hypertension, hypercholesterolemia, and obesity, and complications of diabetes such as myocardial infarction, can lead to cerebrovascular disease and dementia, either alone or in concert with one another (Yaffe, 2007). The published literature as a whole suggests about a two-fold increased risk of stroke in individuals with diabetes, pointing to other pathways also being involved in linking diabetes to dementia.\n\nWhile AD pathology is often cited to be present in about two-thirds of patients with dementia, this estimate includes both patients with AD pathology alone, as well as those with both AD pathology along with other pathologies such as the pathology of VCID and/or other neurodegenerative dementias (e.g., synucleinopathy, TDP-43, etc). Overall, the most common form of dementia is “mixed dementia”, and that mixture is most commonly one of both vascular and neurodegenerative pathology (Schneider et al., 2007). Links between diabetes and AD pathology are also reported but remain poorly understood. Some data from in vivo biomarker studies such as those using neuroimaging and cerebrospinal fluid, suggest that persons with diabetes have more brain atrophy, reduced glucose metabolism, and other changes in markers of neurodegeneration, including alterations in cerebrospinal fluid and phosphorylated tau in particular, that are in keeping with AD (Baker et al., 2011; Moran et al., 2015; Roberts et al., 2014). Mice with knockout of the insulin receptor in the brain show increased tau phosphorylation (Schubert et al., 2004). Data from postmortem brain tissue from elderly individuals have demonstrated the presence of some biochemical features reminiscent of insulin resistance in the hippocampal formation of persons with AD (with and without diabetes) compared to cognitively normal controls, and this insulin resistance has been reported to be associated with pathology that involves both Aβ oligomers and fibrillar amyloid plaques (Talbot et al., 2012). Consistent with this is the report that a mouse model that accumulates only Aβ oligomers showed metabolic flexibility that remained relatively intact while another mouse model that accumulated both Aβ oligomers and fibrils developed features of insulin resistance (Ruiz et al., 2016). A more recent postmortem study has shown that “brain insulin resistance” (a term in need of a precise definition) is associated with tau pathology in AD as well as other neurodegenerative tauopathies such as corticobasal degeneration (Yarchoan et al., 2014). Finally, some genetic data now suggest a link between diabetes and AD, as in the case of the insulin degrading enzyme gene or, more recently the SorCS1 gene which is thought to regulate Aβ metabolism (Lane et al., 2010; Lane et al., 2013). Thus, amyloidosis, tauopathy, typical AD pathology and/or other neurodegenerative mechanisms -- as well as “brain insulin resistance” (perhaps even apart from peripheral diabetes per se) -- appear to play roles in linking diabetes to dementia, apart from the cerebrovascular mechanisms.\n\nNonetheless, neuropathological studies have largely failed to confirm a consistent association between diabetes and AD pathology. Indeed, only a few studies suggest associations with increased AD pathology, including in subsets of persons such as those who are apolipoprotein E epsilon 4 carriers (Malek-Ahmadi et al., 2013; Matsuzaki et al., 2010; Peila et al., 2002), but most studies show either no relationship or a relationship with decreased pathology that is modulated by antidiabetic therapies (Ahtiluoto et al., 2010; Alafuzoff et al., 2009; Arvanitakis et al., 2006a; Beeri et al., 2005; Heitner & Dickson, 1997; Janson et al., 2004; Nelson et al., 2009; Sonnen et al., 2009). There are reports that the clinical cognitive syndrome in diabetes is more dysexecutive rather than amnestic (Arvanitakis et al., 2004; Arvanitakis et al., 2006b; Nandipati et al., 2012), arguing against a strong diabetes-AD connection. In the largest clinicopathological study to date of about 2,400 persons, diabetes was not associated with AD pathology when assessed using overall measures of AD and more specific measures of severity of tangle and amyloid pathology (Abner et al., 2016). Whether some individual features of AD pathology (e.g., Aβ oligomer and/or fibril accumulation) play roles remains unclear. In summary, while an AD mechanism is less established (or perhaps less clinically important) than the vascular mechanism, further elucidation of this pathway may bring insight into other opportunities for treatment and prevention of neurocognitive dysfunction and dementia. Along these lines, both observational studies and experimental clinical trials have examined the role of conventional and unconventional (e.g., intranasal) administration of anti-diabetes medications for treatment of clinical and pathologic AD (Beeri et al., 2008; Craft et al., 2012; Gold et al., 2010; Watson et al., 2005).\n\nYet other mechanisms underlying the association of diabetes with neurocognitive dysfunction and dementia need to be further explored. While not a widely accepted concept, some researchers suggest that some AD may even be considered a metabolic disease caused by insulin resistance in the brain, separate from diabetes (Rivera et al., 2005), raising yet again the need for an evidence-based definition for “brain insulin resistance”. Given the range of effects of insulin in the brain, including neurogenesis, neurite outgrowth, and modulation of catecholamine release/uptake, other possible links between insulin resistance and dementia need to be considered. For examples, mice lacking insulin signaling in brain have been shown to have altered dopamine turnover associated with behavioral changes (Kleinridders et al., 2015). Downstream effects from insulin resistance, whether peripheral or central, could involve increased glucose and advanced glycation end products (AGEs), and alterations in adipokines (e.g., leptin) and other factors (Cherbuin et al., 2012; Holden et al., 2009; Yaffe et al., 2011). Effects of insulin and other factors, including inflammation in the brain, need further consideration as plausible underlying mechanisms linking insulin resistance, with and without diabetes/pre-diabetes, to dementia.\n\nEven in the absence of vascular and other complications, diabetes can alter synaptic plasticity in the mouse hippocampus resulting in cognitive deficits (Stranahan et al., 2008), and mice with diabetes are less likely to recover from stroke due to impaired neuroplastic mechanisms (Sweetnam et al., 2012). Ongoing studies in humans support the hypothesis that cognitive dysfunction in type 2 diabetes is related to alterations in the mechanisms of cortical brain plasticity (Fried et al., unpublished observations). The mechanisms for altered synaptic plasticity in diabetes are unclear but may be linked to reductions in efficacy of N-methyl-D-aspartate receptor (NMDAR)-dependent mechanisms of plasticity. Diabetes is associated with upregulation of the GLUT1 glucose transporter (Kumagai et al., 1996); glucose provides the original source of glutamate in the brain (Bradford & Thomas, 1969), and chronic hyperglycemia may thus lead to excessive glutamate and increased risk of excitotoxicity. Any downregulation in post-synaptic NMDARs to moderate this risk would consequently reduce the efficiency of long-term potentiation and alter any NMDAR-dependent measures. Alterations in the efficacy of the mechanisms of plasticity have also been identified as early pathophysiologic steps in Alzheimer’s disease and these may thus represent another link between metabolic disorders and dementia.\n\nStudies of the relationships linking diabetes, insulin resistance, and neurocognitive dysfunction have largely focused on how peripheral metabolism impacts brain function. Unlike other complications of diabetes (e.g., neuropathy, retinopathy, nephropathy), neurocognitive complications of diabetes have not clearly been demonstrated to correlate with measures of peripheral glycemia (with the exception of very poor glycemic control, HbA1c > 10% and a potential role for glycemic variability) and there is only limited evidence for a modest association with other measures of peripheral glucose regulation (e.g., insulin concentration, insulin action, insulin resistance) (Geijselaers et al., 2015). It is possible that there are central mechanisms that will better account for the neurocognitive dysfunction observed in diabetes and other metabolic disease. One promising line of research has central insulin and insulin-like growth factor 1 (IGF-1) in the spotlight. There is evidence that disrupted central insulin and IGF-1 signaling may lead to disrupted neurotransmitter (e.g., dopamine) and astroglial cell function, brain endothelial cell function involved in formation and regulation of blood-brain barrier (BBB), mitochondrial metabolism and oxidative stress, regulation of the phosphorylation of microtubule-associated tau protein and clearance of Aβ and/or amyloid fibrils, cholesterol synthesis in the brain (important for myelination and membrane function), glucose and lipid metabolism in select regions of the brain, and regulation of central energy balance, which could relate to both metabolic and neurocognitive dysfunction (Bingham et al., 2002; Brüning et al., 2000; Convit et al., 2003; Kleinridders et al., 2015; Liu et al., 2013; Montagne et al., 2015; Schubert et al., 2004; Stouffer et al., 2015; Suzuki et al., 2010; Suzuki et al., 2013). Restoring insulin function in the brain via intranasal insulin is now being tested as a potential therapeutic option for neurocognitive dysfunction in both diabetes and dementia separately (Claxton et al., 2015; Novak et al., 2014) and repurposing diabetes drugs for use in dementia has also been proposed (Yarchoan & Arnold, 2014). However, more research in this area is critically needed. For example, it has been suggested that the reduced insulin signaling observed in aging, and exacerbated in AD, results from a neuroprotective mechanism (Steculorum et al., 2014). This view derives from evidence that decreased insulin signaling promotes life-extension in C. elegans (Kenyon et al., 1993) and that genetically induced insulin resistance has positive effects on cognition in an animal model of AD (Killick et al., 2009). If so, then intranasal insulin treatment may produce short term benefits but deleterious effects in the long term.\n\nThe physiological neuroendocrine mechanism(s) that may participate in the intersection between diet, obesity, diabetes, and dementia is less clear; however, there are a few intriguing mechanisms to consider. Insulin modulates synaptic plasticity of dopamine neurons to regulate reward function and food intake behavior (Stouffer et al., 2015), as well as behaviors of anxiety and depression (Kleinridders et al., 2015) and hyperinsulinemia may lead to ineffective regulation of dopamine and increased food intake (Liu et al., 2013). It is also possible that hormonal changes that occur in obesity and diabetes, such as disrupted glucocorticoid function, could impact synaptic plasticity and brain function (Bocarsly et al., 2015). Impaired neuroplasticity itself, via a glucocorticoid-related or other mechanism(s), could account for metabolic and neurocognitive dysfunction (Oberman & Pascual-Leone, 2013; Pascual-Leone et al., 2011; Stranahan et al., 2008; Wosiski-Kuhn et al., 2014). There is also evidence that corticotropin-releasing factor regulates cerebral amyloid accumulation along an as yet undefined pathway that is glucocorticoid-independent (Macauley et al., 2015).\n\nMicroglial dysfunction has become a central point for investigation in AD, since about one-third of genes associated with AD are expressed at high levels or exclusively by microglia. Glucocorticoids appear to impact microglia activation and the release of pro-inflammatory cytokines, which may result in neuroinflammation and cognitive dysfunction (Dey et al., 2014; Erion et al., 2014; Hao et al., 2016). Somewhat unexpectedly, the gut microbiome has been recently revealed as a potential modulator of physiological function of microglia, and research in other neurodegenerative diseases and Parkinson Disease in particular, have suggested a gut-brain mechanism of disease (Erny et al., 2015).\n\nThere has been even less appreciation for how diet and obesity may adversely impact brain function in the absence of diabetes or dementia. Obesity has been associated with altered brain structure and function in animal models and in metabolically and neurologically healthy adults and children (Bocarsly et al., 2015; Hsu & Kanoski, 2014; Yau et al., 2014). Consumption of a high fat diet (HFD) can also negatively impact the brain and cognitive function well before obesity onset (Hsu & Kanoski, 2014). Indeed, obesity can impact cognition independently from metabolic disease and diet can impact metabolic function and cognition independently of obesity. Recent data suggest that weight reduction may even improve neurocognitive function (Horie et al., 2016). It will therefore be important to look beyond disease states to examine the influence of diet and obesity on neurocognitive function more generally.\n\nAlthough obesity is occasionally associated with global measures of brain atrophy (Brooks et al., 2013; Enzinger et al., 2005; Raji et al., 2010) and cognitive decline, many studies suggest that executive function and learning and memory are most affected in both adults and children (Fitzpatrick et al., 2013; Francis & Stevenson, 2013; Gunstad et al., 2007; Sabia et al., 2009). Corresponding with this neuropsychological profile, structural changes are observed in the parietal and prefrontal cortex (important for executive function), as well as the entorhinal cortex and hippocampus (important for learning/memory) (Enzinger et al., 2005; Fotuhi et al., 2012; Hao et al., 2016; Miller & Spencer, 2014; Pannacciulli et al., 2006; Raji et al., 2010). These changes may be a consequence rather than a cause of obesity, as supported by neuroimaging data in the minipig animal model (Val-Laillet et al., 2011). Cerebral blood flow, a marker of neuronal activity, is significantly lower in the dorsolateral and anterior prefrontal cortex in minipigs with diet induced obesity compared to their lean counterparts. Likewise, in humans, there is reduced cortical thickness in the prefrontal, temporal and parietal cortex in adults but not in children (Sharkey et al., 2015), suggesting that structural changes occur after chronic HFD, metabolic disease and/or obesity. Teasing apart these three factors is an important avenue for future work.\n\nHFD and obesity are also associated with dopamine-dependent mesocorticolimbic-prefrontal alterations that may impact reward learning, motivation, and executive functions (Johnson & Kenny, 2010; Sevgi et al., 2015; Stice et al., 2008; Sun et al., 2015; Vainik et al., 2013). Overweight/obese compared to healthy weight individuals show reduced change in striatal D2R binding potential in response to glucose ingestion (consistent with reduced dopamine release) and several studies have reported a negative association between basal metabolic index and the blood oxygen level dependent (BOLD) response to milkshake consumption in the dorsal striatum. Although BOLD does not directly measure dopamine release, the effect is dependent upon the Taq1a A1 polymorphism, which affects D2 receptor density, thus linking the BOLD response to abnormal D2R signaling which can also be linked to heightened impulsivity (Babbs et al., 2013). Along this line, there are emerging data that suggest specific macronutrients or combinations of macronutrients have selective effects on dopamine adaptations. One can speculate on the development of “vicious cycles” that lead to obesity, driven by both genetic makeup, dietary composition, and other factors.\n\nSimilar to the association between diabetes and AD, “brain insulin resistance” may play a key role. Insulin action directly affects central glucose metabolism and is posited to influence learning and memory by modulating synaptic plasticity, density, neurotransmission and adult neurogenesis. As such, intranasal administration improves cognition in healthy individuals (Benedict et al., 2004), raising the possibility that the reverse is also true; that is, decreased insulin signaling associated with sub-clinical insulin resistance accounts for the cognitive dysfunction observed following a HFD or obesity in otherwise healthy individuals. Supporting this hypothesis, a functional type of “brain insulin resistance” has been associated with hypo-active responses in obese non-diabetic, cognitively intact, people within fronto-parietal circuits, which are critical for working memory (the ability to hold and manipulate new and old information “on-line”). Working memory is markedly impaired in overweight and obese individuals and insulin sensitivity mediates a negative association between body mass index (BMI) and response in the fronto-parietal circuit during a working memory task (Gonzales et al., 2010). Diet-induced obesity can also influence insulin resistance in the hippocampus and likely elsewhere in the brain where insulin receptors are expressed, such as the amygdala and midbrain (McNay et al., 2010). Thus, metabolic alterations may impact brain function well before the onset of metabolic disorders, and weight reduction may improve both metabolic state and cognition (Horie et al., 2016).\n\nTypical peripheral insulin resistance is often correlated with a number of other factors associated with diet-induced obesity that may also cause neurocognitive dysfunction. Obesity and HFD lead to systemic and central inflammation with elevated circulating IL-12 and IL-6, both of which have been linked to impaired processing speed and executive function, even independently of metabolic risk factors (Gregor & Hotamisligil, 2011; Thaler & Schwartz, 2010; Trollor et al., 2012). Thus, several groups have highlighted inflammation as “the pathway to cognitive impairment” (Miller & Spencer, 2014; Steculorum et al., 2014). Hypersecretion of glucocorticoids may also contribute. The hypothalamic-pituitary-adrenal axis, which is altered in obesity, regulates glucocorticoid secretion, and has in turn been associated with mood changes, memory impairment, and reduced hippocampal volume (MacQueen & Frodl, 2011; Raber, 1998).\n\nEmerging work also suggests that diet-induced BBB disruption may provide a mechanistic link involving diet, obesity and hippocampal-dependent cognitive functions (Hsu & Kanoski, 2014). Rats fed a HFD for 90 days show decreased expression of BBB tight junction proteins and the presence of sodium fluorescin, a fluorescent-tagged molecule normally excluded from the brain by the BBB and choroid plexus, was found in the hippocampus but not in the prefrontal cortex or striatum (Kanoski et al., 2010). More recently, Davidson and colleagues found sodium fluorescin in the hippocampus of rats that failed to show a reversal of diet-induced impairments in hippocampal-dependent tasks upon HFD discontinuation, while rats demonstrating recovery of function did not (Davidson et al., 2012). Some evidence indicates that BBB integrity may influence learning and memory by affecting the passage of nutrients and neuroendocrine signals such as insulin, leptin and ghrelin from the periphery to the brain.\n\nMany of the brain adaptations produced by diet, obesity and metabolic dysfunction lead to neurocognitive and behavioral changes that may confer additional risk for obesity (Higgs et al., 2012; Higgs, 2015a; Higgs, 2015b; Sellbom & Gunstad, 2012; Vainik et al., 2013), leading to what has been described as a “vicious cycle of obesity, metabolic disease, and cognitive decline” (Davidson et al., 2014). Insensitivity to satiety can disrupt habituation or the drop in appetitive responses to foods that accompany eating and there is evidence that the hippocampus plays a role (Small et al., 2001). Work in rodents has also focused on the role of the hippocampus in sensing the internal state. In an associative learning model, diet-induced damage to the hippocampus may disrupt the ability of interoceptive satiety states to serve as contextual stimuli signaling that a food is not rewarding in a sated state (Davidson et al., 2014). This, in turn, leads to positive energy balance by promoting eating in the absence of hunger.\n\nThe decreased responses in fronto-parietal circuits and corresponding executive function impairments have long been implicated in inhibitory control of feeding behavior (Alonso-Alonso & Pascual-Leone, 2007; Le et al., 2006). Adaptations in these circuits may therefore further promote overeating, especially in the modern food environment where self-control is especially taxed. In addition, decisions to eat depend upon multiple interacting factors besides hunger and hedonics. Habits, time of day, heath concerns, peer pressure, and stress are amongst the other important modulators. The prefrontal cortex is critical for integrating these factors so that long-term goals can override hedonic and homeostatic signals.\n\nThus, while strong evidence links obesity to brain dysfunction, the extent to which this arises directly from adiposity, diet, metabolic dysfunction, or other factors is incompletely understood and future research in this area is needed.\n\n\nSome research areas in need of increased attention\n\nThe role of early development, in particular, appears to be critical in understanding the link between obesity, metabolic disease, and neurocognitive dysfunction. Maternal obesity and metabolic disease, and diet during pregnancy may impact the child in utero and lead to a greater risk for childhood obesity, metabolic dysfunction, and neurodevelopmental disruption (Linder et al., 2015; Rivera et al., 2015). High-fat feeding of pregnant and nursing mice has been shown to have important effects of hypothalamic neurocircuit formation that impacts adult brain function (Vogt et al., 2014). Intervention in early development is critical as there is now evidence that dysregulated metabolic function and obesity lead to detectable brain and cognitive changes in young children, may disrupt academic performance, and could lead to challenges in transitioning to independence and successful adulthood (Mauras et al., 2015; Mazaika et al., 2016; Semenkovich et al., 2015; Yau et al., 2012; Yau et al., 2014).\n\nWhile the association between type 2 diabetes and dementia has been reported for some time, there has been less attention paid to whether type 1 diabetes may be associated with dementia. We now know that childhood-onset type 1 diabetes leads to detectable brain and cognitive changes in early life through middle adulthood (Mauras et al., 2015; Mazaika et al., 2016; Nunley et al., 2015a; Nunley et al., 2015b; Ryan et al., 2015; Semenkovich et al., 2015), including changes in brain fMRI (Musen et al., 2008). It will be important to understand how glycemic dysregulation over the lifecourse impacts neurocognitive function, disease management, and risk for dementia. Iatrogenic hypoglycemia may play some role(s) that remain to be clarified.\n\nA complex and confusing literature has already accumulated wherein no obvious single, simple model adequately explains the bidirectional relationships linking central and peripheral insulin signaling and metabolism. Because of this, we are not attempting to summarize all that literature here. However, we would refer the reader to Ishii & Iadecola (2015b), (Ruiz et al., 2016), and (Knight et al., 2016). The former provides an outstanding summary of the primary and secondary involvement of the hypothalamus in AD, while the latter two provide examples of the largely unappreciated metabolic dysfunction present in standard models of AD pathology and the unexpected effects that can result when either the AD pathology, the metabolic pathology, or both, are manipulated.\n\n\nSome broad research needs\n\nThe workshop concluded with discussion of the research presented and reviewed above in order to identify research needs to move the field forward. Below is a list of the most important research needs identified at the workshop.\n\n• Improved tools and model systems for drug discovery and development\n\n• Increased utilization of existing tools and model systems for mechanistic research\n\n• Deep metabolic and neurocognitive phenotyping in human studies\n\n• Improved biomarkers for use in human studies\n\n• Increased utilization of existing biomarkers for human studies\n\n• Improved human neuroimaging technologies\n\n• Detailed metabolomics analysis of individual brain regions in both diabetes and dementia, using techniques such as imaging mass spectrometry\n\n• Increased utilization of existing, novel human neuroimaging technologies\n\n• Longitudinal human studies assessing cognition, metabolism and diet\n\n• Human studies manipulating diet to determine causality and identify specific aspects of foods (e.g. macronutrients such as saturated fatty acids) that negatively impact brain and brain function\n\n• An operational definition of “brain insulin resistance” and tools for measuring brain insulin signaling in vivo\n\n• Better understanding of insulin action on astroglial versus neuronal cells as this relates to altered brain function in diabetes\n\n• Development of a protocol to define and measure a “brain health index” for persons with metabolic dysfunction\n\n• Need for harmonization and standardization across measures and protocols\n\n• Combined human, animal, and cell culture studies\n\n• Increased attention to gut-brain signaling, microbiome, and the relationship to obesity, metabolic disease, and neurocognitive dysfunction\n\n• Evaluation of weight loss and diet for reversing or improving cognition\n\n• Interdisciplinary teams collaborating on research at the intersection of metabolism and brain dysfunction\n\n• Engaging and maintaining junior researchers in the field\n\n\nFuture directions\n\nNow is a critical period for research in the field of metabolic and neurocognitive dysfunction. Major complementary efforts, including the Brain Research Through Advancing Innovative Neurotechnologies (BRAIN) initiative, the National Plan to Address Alzheimer’s Disease (NAPA), the Big Data to Knowledge (BD2K) initiative, and the Precision Medicine Initiative (PMI), will provide the financial support, tools, and scientific communities that are poised to substantially advance mechanistic research linking metabolic disease and neurocognitive dysfunction within the next dozen or so years. The scientific challenge here is to determine the mechanism(s) that explain the interactions between metabolism, the brain, and neurocognitive dysfunction, in order to identify methods for early detection of risk for metabolic and neurocognitive dysfunction and potential strategies and critical periods for prevention, mitigation, and intervention for these diseases. Figure 1 illustrates the most plausible biological mechanisms that may explain the bidirectional relationship between metabolic and neurocognitive dysfunction. These include mechanisms at the molecular, cellular, organ and tissue-based, and systemic levels, which may lead to metabolic and neurcognitive dysregulation that underlies disease processes contributing to obesity, insulin resistance, prediabetes, type 2 diabetes, and dementia (including VCID, VaD, AD, and others). In addition, these factors may be modified by other biological, behavioral, and environmental factors, which may be targets to optimize health outcomes. Public policy can serve as the ultimate modifier of these various risk factors, and will determine the success of the major effort required to prevent these common co-morbid diseases that are increasingly likely to burden our aging population.\n\nAn important step toward that goal is to take a highly heterogeneous group of individuals with metabolic and neurocognitive dysfunction and begin to cluster them into more homogeneous subgroups based on detailed clinical, metabolic, and neurocognitive data. This includes the need for more human brain specimens from individuals with good physiological characterization regarding diabetes, metabolic function, and neurocognitive function throughout the lifespan. This would allow preclinical researchers to test potential mechanisms based on distinct, relevant metabolic and neurocognitive abnormalities and develop prevention and treatment strategies targeting these phenotypes. Animal models will be an important contribution to these preclinical studies and should have comparable abnormalities in conserved domains compared to humans. For example, in rodents, it is reasonable to assume that learning and memory functions are somewhat comparable to humans, but it is unlikely that executive functions and decision-making (and the neural circuits that support these functions) are similar between species. Finally, a more homogeneous cluster of individuals with shared metabolic and neurocognitive resilience, risk or disease factors are allocated to targeted prevention and intervention efforts based on shared mechanisms to optimize prevention, mitigation, and treatment of the neurocognitive complications of diabetes and related metabolic dysfunction. In many ways, we already have the tools and technologies in place to perform deep phenotyping to guide future efforts for targeted, individualized therapies. Recently, it was discovered based on genetic analyses, that type 2 diabetes may in fact consists of 3 subtypes and that one specific subtype appears to be associated with neurological disease (Li et al., 2015). This type 2 diabetes subtype was also associated with cardiovascular disease, but not diabetic retinopathy or nephropathy, which implies that the mechanisms for neurocognitive complications of diabetes differ from complications in other end organs. There are related efforts, methods, and tools in development that could be leveraged for further discovery into the mechanisms at the interface of metabolic and neurocognitive function, including in the areas of neuroimaging (Yeo et al., 2016) and neuroimaging genetics (Thompson et al., 2014), web-based cognitive testing (Germine et al., 2012), and individualized neural and physiological phenotyping over time (Poldrack et al., 2015; Wang et al., 2015; Yeo et al., 2016).",
"appendix": "Author contributions\n\n\n\nL.E.S., Z.A., S.G., and D.S. wrote the first draft, revised, and edited the White Paper. Other named authors read and edited the paper. All authors approved the content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe following National Institutes of Health grant funding supported this work: R01 NS084965 (Z.A.); U01AG046170 (S.G.; Eric Schadt); R01 DK 085579 (D.S.); P50 AG005138 (S.G.; Mary Sano, PI); R01 CA 180030 (D.S.); R01 NS075685 (S.G.); R01 DC 006706 (D.S.); P30 AG10161 (Z.A. David A. Bennett, PI); R01 AG 040039 (Z.A.); R21 NS082870 (A.P.L.).\n\n\nAcknowledgements\n\nThis White Paper was submitted on behalf of the participants of the National Institute of Diabetes, Digestive, and Kidney Diseases Workshop “The Intersection of Metabolic and Neurocognitive Dysfunction” in July 2015. For details of the workshop program, see the Supplementary File 1. Thank you to Mr. Alan Hoofring (Lead Medical Illustrator, NIH) for his graphics expertise in the creation of Figure 1.\n\n\n\n\nThe most plausible biological mechanisms (middle panel: molecular, cellular, organ and tissue-based, systemic) are shown that may explain the bidirectional relationship between metabolic (left panel: obesity, insulin resistance, prediabetes, type 2 diabetes) and neurocognitive dysfunction, including dementia (right panel: e.g., VCID, VaD, AD, other). Genetic and epigenetic mechanisms (not represented in the figure) likely trigger the pathophysiological processes underlying metabolic and neurocognitive dysfunction (represented by the innermost circle), and these disease processes, in turn, may be modified by other factors (listed under the “policy” umbrella), which may be targets to maximize health outcomes and behaviors. 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}
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[
{
"id": "12962",
"date": "12 Apr 2016",
"name": "Steven W Barger",
"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 rather comprehensive review of the connections between the metabolic syndrome associated with Type-2 diabetes mellitus (T2DM) and cognitive impairment was inspired by a workshop held by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). The article is so thorough and well organized that one assumes the proceedings of that meeting must have been only the starting point and that the thoughts and opinions arising from that brainstorming session have been embellished with considerable diligence and reflection here. Most of the points covered in this “white paper” are well reasoned and well researched. And all the topics represent elements of pathology and pathogenesis that will be key to any faithful attempt to understand the intersection between T2DM, dementia, and milder forms of cognitive impairment. Other authors have often succumbed to the temptation to focus on Alzheimer’s disease (AD) as the tag-word for these discussions, but one of the triumphs of this review is its more general consideration of dementia and sub-demented deficiencies as per their connections to energy substrates, both in the central nervous system and peripherally. Indeed, the authors highlight a consideration that seems to be gaining in prominence and frequency in discussions of AD itself: the fact that heterogeneity—especially within certain classifications of age, gender, and genetics—suggests not only multiple routes to AD, but also multiple disease endpoints. Particularly within the “oldest-old” populations, APOE4-negative individuals, and high-risk populations such as military veterans, classic AD pathology correlates rather loosely with cognition. To their credit, the authors discuss the possibility that metabolic derangement may yet be shown to be the driving force for at least some of these cases: \"[I]t is still unclear if the relationship between diabetes and neurocognitive dysfunction and dementia is due to classic AD pathology, vascular processes such as infarcts or vessel pathology, other pathologies less closely related to AD or vascular disease (e.g., impaired brain insulin signaling), or some combination of these. Some have proposed that ‘all-cause’ dementia be employed in order to avoid the implication that we can classify etiologies accurately based on current knowledge.\" Another important point made here regards the direction of causality. Hypotheses inspired by the earliest epidemiological links between dementia and T2DM presumed that complications of the latter drive the neuropathologies (mostly, those related to protein aggregation) that underlie AD and related dementias. But a more accurate picture of the relevant events in AD progression has been afforded—largely through advances in neuroimaging tools—and this insight sets the stage for researchers to now say, \"…the pathophysiological processes that lead to metabolic and neurocognitive dysfunction precede the development of clinical syndromes by years…. Indeed, AD neuropathology is detectable more than 10 years before the onset of the first clinical symptoms (Bateman et al., 2012) and pre-diabetes also precedes diabetes by years in most cases.\" In fact, data indicate that changes in amyloid β-peptide (Aβ) precede reductions in cerebral glucose utilization, the latter of which may be an important index of metabolic syndrome (below). Supported by evidence from animal models, emerging trends in hypothesis-building include the supposition that CNS accumulation of Aβ, and perhaps other elements of neurodegeneration, are sufficient to perturb peripheral metabolism. This allows consideration of the possibility that correlations between cognitive impairment and cortical amyloid burden are so capricious because an important modulatory (or even instigating) factor is the accumulation of Aβ in subcortical regions: \"Therefore, it is plausible that AD pathology (amyloid-beta or Aβ, tauopathy) may be present in the hypothalamus prior to the onset of cognitive symptoms, and that this hypothalamic pathology could disrupt homeostatic functions such as energy balance and peripheral metabolism prior to the onset of detectable cognitive dysfunction (Ishii & Iadecola, 2015b).\" It is also encouraging to see the inclusion of information on effects of obesity and diet that appear to be independent of glycemic regulation. This is an intriguing body of data, considering the untoward effects of high-fat diets in rodents vis-à-vis the benefits that ketogenic diets and their mimics seem to have in humans 1. It seems that metabolic impacts on brain health could benefit from better understanding of the effects that are brought to bear by shifts between the prominent energy-substrate molecules, perhaps through impacts on such substances that also impact neurotransmitter pools, such as glutamate and glutamine. And it might be productive here to consider hypotheses regarding the finding that obesity in midlife is correlated with dementia manifest decades later 2, at a time when weight loss may be more common. For all the salient points made in this white paper, the authors still find themselves perpetuating a conventional assumption that seems shortsighted in light of a substantial body of evidence; this is all the more disappointing because they consider what the implications would be “if metabolic disease impacts the brain in ways that differ from its effects on other end organs impacted by diabetes and other metabolic disease.” This is almost certainly the case. The traditional view that elevated glucose in the blood must, by necessity, produce elevated glucose in the brain ignores the existence of the blood-brain barrier (BBB); very little glucose can enter the cerebrum, or most other regions of the CNS, without regulated transport, the bulk of which is handled by glucose transporter 1 (GLUT1). Thus, it was quite surprising to read a major conclusion in the paper that seems to have been based primarily on a single publication, and moreover, one devoted not to the brain but to the retina: \"Diabetes is associated with upregulation of the GLUT1 glucose transporter (Kumagai et al., 1996); glucose provides the original source of glutamate in the brain (Bradford & Thomas, 1969), and chronic hyperglycemia may thus lead to excessive glutamate and increased risk of excitotoxicity.\" This is subject to criticism on two counts: 1) Several studies show that AD and hyperglycemia are both associated with reductions in endothelial glucose transporters, likely reflecting—at least in the latter—a reactive feedback inhibition; this probably contributes to the diminished 2-deoxyglucose accumulation in both disorders. 2) Excitotoxicity is more likely to be a consequence of glucose deprivation; this is a well-established relationship 3,4, whereas the claim that high glucose elevates glutamate release is tenuous and unsupported by citations. Publications documenting a diminution of GLUT1 levels in hyperglycemia and/or the converse, an elevation in hypoglycemia, comprise an overwhelming body of evidence 5-10. The authors later provide some evidence suggesting effects on the BBB from metabolic abnormalities in rat models, particularly those brought on by diet, but data in humans is spotty and of unclear relevance to glucose delivery. Less serious or less consequential issues arose with regard to a few other elements in the paper: \"Unlike other complications of diabetes…, neurocognitive complications of diabetes have not clearly been demonstrated to correlate with measures of peripheral glycemia (with the exception of very poor glycemic control, HbA1c > 10% and a potential role for glycemic variability) and there is only limited evidence for a modest association with other measures of peripheral glucose regulation….\" Why would it be necessary—or indeed, even helpful—to exclude HbA1c levels and glycemic control from the calculus? Which indices of peripheral glycemia would be more justifiable? To be sure, 10% is a quite high level for HbA1c, but lower levels might show a detectable effect in an adequately powered study. Moreover, HbA1c might not change dramatically under conditions of highly fluctuating blood-glucose concentrations, and yet such wild swings could easily produce intolerable strains on glucose-sensitive tissues—including brain endothelium—simply by virtue of their volatility. The authors are to be applauded for their mention (however brief) of the apparent paradox between the ill effects of pathological insulin resistance versus the benefits of experimentally interrupting insulin signaling, at least in C. elegans. It should be noted that similar benefits (e.g., on live span) accrue in mammals from inhibition of signaling for insulin-like growth factor (IGF), and C. elegans have only one peptide to accomplish the roles of both insulin and IGF. Nevertheless, the conundrum is one that has vexed experimental gerontologists for decades, owing in part to the similar benefits afforded by attention of the insulin/IGF signaling (IIS) pathway and by dietary restriction, the latter of which certainly reduces overall insulin production. One should not expect the authors to have cracked this tough nut, but important rumination might result from a slightly more complete discussion of the differences between reactive (feedback-mediated) insulin resistance and the proactive sort effected by empirical manipulation of IIS. Also given short shrift here is the undeniable connection of inflammation to both metabolic syndrome and neurocognitive disorders. This is obviously an underappreciated link that deserves greater attention among scientists seeking to synthesize these fields; reviewers of grants and papers continue to express befuddlement when the topic of neuroinflammation is broached in this context. Given the overwhelming evidence for inflammation-related events in the etiologies of both T2DM and AD, it is almost certain to contribute to their coincidence. In fact, compelling evidence has supported just this sort of causal involvement in animal models 11. The authors have included the word “inflammation,” distributed diffusely in the paper; perhaps it would create the appropriate emphasis if some of these references were consolidated in one or two paragraphs along with more specific information, e.g., the seminal role of IKK2 in insulin resistance 12 and Aβ effects 13. Finally, the paper’s conclusion with “Some broad research needs” is doubtlessly faithful to the output of the workshop’s deliberations, but many of the points outlined in this list come across as rather vague. Without further elaboration, it is not clear what gaps in knowledge some of them would address. One wonders if several could be combined and embellished with a bit more elaboration. In its overall contribution, this white paper provides a very useful and much-needed synthesis of data and concepts regarding the connections between cognition and metabolic derangement. Modifiable elements of lifestyle and environment have recently emerged as major considerations in the prevention of dementia. Stoeckel et al. have enhanced the conversation by broadening it to other categories of cognitive impairment. In addition, they largely succeeded in summarizing the current state of fields that can seem disparate, and they highlight opportunities to draw these fields closer.",
"responses": [
{
"c_id": "2008",
"date": "02 Jun 2016",
"name": "Luke Stoeckel",
"role": "Author Response",
"response": "Referee Reports: 12 April 2016 (SW Barger): We appreciate that Dr. Barger felt this review was “so thorough and well organized that one assumes the proceedings of that meeting must have been only the starting point and that the thoughts and opinions arising from that brainstorming session have been embellished with considerable diligence and reflection here.” We also thank the referee for concluding that “In its overall contribution, this white paper provides a very useful and much-needed synthesis of data and concepts regarding the connections between cognition and metabolic derangement. Modifiable elements of lifestyle and environment have recently emerged as major considerations in the prevention of dementia. Stoeckel et al. have enhanced the conversation by broadening it to other categories of cognitive impairment. In addition, they largely succeeded in summarizing the current state of fields that can seem disparate, and they highlight opportunities to draw these fields close”. Point 1: “It is also encouraging to see the inclusion of information on effects of obesity and diet that appear to be independent of glycemic regulation. This is an intriguing body of data, considering the untoward effects of high-fat diets in rodents vis-à-vis the benefits that ketogenic diets and their mimics seem to have in humans 1. It seems that metabolic impacts on brain health could benefit from better understanding of the effects that are brought to bear by shifts between the prominent energy-substrate molecules, perhaps through impacts on such substances that also impact neurotransmitter pools, such as glutamate and glutamine. And it might be productive here to consider hypotheses regarding the finding that obesity in midlife is correlated with dementia manifest decades later 2, at a time when weight loss may be more common.” Response: Text modified to make this important point clear (see p. 6, paragraph 1). “Compared to adults with normal body mass index (BMI = 18.5 – 24.9), overweight and obese adults in midlife (ages 40-59 years) have a relative risk (RR) of 1.35 (overweight) and 2.04 (obese) for AD and 1.26 (overweight) and 1.64 (obese) of any cause dementia (Anstey et al. 2011). This association between overweight and obesity is not observed in late life (ages 60 years or older) at a time when weight loss is more common and a risk factor for dementia (Anstey et al. 2011).” Point 2: “For all the salient points made in this white paper, the authors still find themselves perpetuating a conventional assumption that seems shortsighted in light of a substantial body of evidence; this is all the more disappointing because they consider what the implications would be “if metabolic disease impacts the brain in ways that differ from its effects on other end organs impacted by diabetes and other metabolic disease.” This is almost certainly the case. The traditional view that elevated glucose in the blood must, by necessity, produce elevated glucose in the brain ignores the existence of the blood-brain barrier (BBB); very little glucose can enter the cerebrum, or most other regions of the CNS, without regulated transport, the bulk of which is handled by glucose transporter 1 (GLUT1). Thus, it was quite surprising to read a major conclusion in the paper that seems to have been based primarily on a single publication, and moreover, one devoted not to the brain but to the retina: \"Diabetes is associated with upregulation of the GLUT1 glucose transporter (Kumagai et al., 1996); glucose provides the original source of glutamate in the brain (Bradford & Thomas, 1969), and chronic hyperglycemia may thus lead to excessive glutamate and increased risk of excitotoxicity.\" This is subject to criticism on two counts: 1) Several studies show that AD and hyperglycemia are both associated with reductions in endothelial glucose transporters, likely reflecting—at least in the latter—a reactive feedback inhibition; this probably contributes to the diminished 2-deoxyglucose accumulation in both disorders. 2) Excitotoxicity is more likely to be a consequence of glucose deprivation; this is a well-established relationship 3,4, whereas the claim that high glucose elevates glutamate release is tenuous and unsupported by citations. Publications documenting a diminution of GLUT1 levels in hyperglycemia and/or the converse, an elevation in hypoglycemia, comprise an overwhelming body of evidence 5-10. The authors later provide some evidence suggesting effects on the BBB from metabolic abnormalities in rat models, particularly those brought on by diet, but data in humans is spotty and of unclear relevance to glucose delivery.” Response: We agree with the reviewer and have reorganized the section on “Mechanisms linking diabetes to dementia (p. 3)” and have modified discussion of the relationship between peripheral hyperglycemia and CNS hyperglycemia, GLUT1 glucose transporter, and excitotoxicity in response to the reviewer’s feedback. Specifically, we have indicated that excitotoxicity in the brain may be more closely related to glucose deprivation via reductions in endothelial glucose transporters. The following text can be found on p. 4, paragraph 3: “Chronic hyperglycemia has been associated with reductions in endothelial glucose transporters such as GLUT1 that could lead to glucose deprivation and excitotoxicity, and could account for some of the neurocognitive complications of diabetes (Gjedde and Crone 1981; Matthaei et al. 1986). This is unlike diabetic retinopathy, which has been associated with upregulation of GLUT1 glucose transporter ( Kumagai et al., 1996) and is another example of how diabetic complications in the brain may differ from other organ systems.” Point 3: \"Unlike other complications of diabetes…, neurocognitive complications of diabetes have not clearly been demonstrated to correlate with measures of peripheral glycemia (with the exception of very poor glycemic control, HbA1c > 10% and a potential role for glycemic variability) and there is only limited evidence for a modest association with other measures of peripheral glucose regulation….\" “Why would it be necessary—or indeed, even helpful—to exclude HbA1c levels and glycemic control from the calculus? Which indices of peripheral glycemia would be more justifiable? To be sure, 10% is a quite high level for HbA1c, but lower levels might show a detectable effect in an adequately powered study. Moreover, HbA1c might not change dramatically under conditions of highly fluctuating blood-glucose concentrations, and yet such wild swings could easily produce intolerable strains on glucose-sensitive tissues—including brain endothelium—simply by virtue of their volatility.” Response: We appreciate the need for clarification on this comment. We did not intend to suggest that HbA1c or glycemic variability were unimportant, and we agree that some existing studies may be underpowered to detect effects at lower levels of hyperglycemia (i.e., HbA1c < 10%). We have modified our text to emphasize future studies should not ignore the roles of HbA1c and glycemic variability, and there is a need for more adequately powered studies to clarify the roles for these variables in cognitive impairment and dementia. The following text can be found on p. 5, paragraph 2: “It is possible that lower levels of hyperglycemia (i.e., HbA1c < 10%) may have detectable effects on neurocognitive function in adequately powered studies or that HbA1c levels may not reflect highly fluctuating blood glucose concentrations, which could have independent deleterious effects on glucose-sensitive tissues such as brain endothelium.” Point 4: “The authors are to be applauded for their mention (however brief) of the apparent paradox between the ill effects of pathological insulin resistance versus the benefits of experimentally interrupting insulin signaling, at least in C. elegans. It should be noted that similar benefits (e.g., on live span) accrue in mammals from inhibition of signaling for insulin-like growth factor (IGF), and C. elegans have only one peptide to accomplish the roles of both insulin and IGF. Nevertheless, the conundrum is one that has vexed experimental gerontologists for decades, owing in part to the similar benefits afforded by attention of the insulin/IGF signaling (IIS) pathway and by dietary restriction, the latter of which certainly reduces overall insulin production. One should not expect the authors to have cracked this tough nut, but important rumination might result from a slightly more complete discussion of the differences between reactive (feedback-mediated) insulin resistance and the proactive sort effected by empirical manipulation of IIS.” Response: We appreciate this important point from the reviewer; however, this did not need receive substantial discussion at the workshop and, given the complexity of this issue, would require substantial more attention beyond the scope of the current paper. We have acknowledged this point in the manuscript. The following text can be found on p.5, paragraph 2: “The apparent paradox between pathological insulin resistance and the benefits of acute disruption of insulin signaling is a complex topic requiring further research attention.” Point 5: “Also given short shrift here is the undeniable connection of inflammation to both metabolic syndrome and neurocognitive disorders. This is obviously an underappreciated link that deserves greater attention among scientists seeking to synthesize these fields; reviewers of grants and papers continue to express befuddlement when the topic of neuroinflammation is broached in this context. Given the overwhelming evidence for inflammation-related events in the etiologies of both T2DM and AD, it is almost certain to contribute to their coincidence. In fact, compelling evidence has supported just this sort of causal involvement in animal models 11. The authors have included the word “inflammation,” distributed diffusely in the paper; perhaps it would create the appropriate emphasis if some of these references were consolidated in one or two paragraphs along with more specific information, e.g., the seminal role of IKK2 in insulin resistance 12 and Aβ effects 13.” Response: We agree with the reviewer that this is another important area for further research attention. However, inflammation in the context of diabetes and dementia was not the primary focus of the workshop; therefore, we refer the reader to recent reviews for more in depth attention to this important topic. The following text can be found on p.5, paragraph 1: “Inflammation-related events in the periphery and brain may be another important factor explaining the connection between diabetes and neurocognitive dysfunction (see (Ferreira et al. 2014; De Felice and Lourenco 2015) for recent reviews of this topic).” Point 6: “Finally, the paper’s conclusion with “Some broad research needs” is doubtlessly faithful to the output of the workshop’s deliberations, but many of the points outlined in this list come across as rather vague. Without further elaboration, it is not clear what gaps in knowledge some of them would address. One wonders if several could be combined and embellished with a bit more elaboration.” Response: We apologize for being overly vague in this section and have revised this section to provide more clarity."
}
]
},
{
"id": "13698",
"date": "06 May 2016",
"name": "Mahmoud Reza Azarpazhooh",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 offer a balanced review regarding the relationship between cognitive decline and insulin resistance. They cover a majority of important studies, providing excellent insights into our current knowledge, and more importantly gaps in definition, pathophysiology, and prevention and treatment of cognitive impairment related to diabetes.One of the strongest parts of the paper is highlighting a probable reciprocal cause and effect relation between diabetes, leading to the cognitive decline, and neurodegenerative lesions in the brain, resulting in central insulin resistance. On the one hand, diabetes can contribute to dementia via a range of mechanisms, including vascular components1, peripheral and central inflammatory response via activation of microglia2, change in the blood brain barrier3, and neurostructural changes, both in white and gray matter4. On the other hand, neurodegenerative changes, such as amyloid deposition in the brain, especially in the hypothalamus5, may lead to insulin resistance and probably a central type of metabolic disorders and diabetes. This idea opens a new window in organized research for dementia and diabetes and at the same time underlines an urgent need to clarify the priorities in these vital fields.This paper provides a long list of possible gaps and plans for further studies; however, some themes stand out:An urgent need for definitions, and collaborations: Despite a relatively high incidence of diabetes and dementia and not surprisingly extensive research in these conditions, still no worldwide consensus exists on definitions, inclusion and exclusion criteria and clarity on some key words. This can be partially explained by focused research in diabetes and dementia, conducted in different disciplines in parallel and sometimes opposite directions. Considering diabetes as a systemic disease, affecting almost each and every part of human body, and cognitive decline, as a condition with a wide range of etiologies, and presentations, it is a high time to look at these common disorders in a more holistic, multi-dimensional vision and create data banks, including all available data and use similar definitions, and common vocabularies. Such national/international data banks may provide a better insight into epidemiology, pathophysiology, prevention, and treatment of dementia and diabetes and at the same time can save our limited health resources. Experimental studies: Animal studies play a significant role on our current knowledge in medicine. Despite their undeniable advantages in the evaluation of pathogenesis and treatment of several diseases, they still suffer from specific limitations, mainly due to high costs as well as the selection of the most appropriate animals. A similar significant gap can also be seen in animal studies of dementia and diabetes, using different animals, with limited sample sizes and lack of enough controls. Therefore, it may be worthwhile creating national/ international data banks for animal samples from both sexes with or without diseases, covering all possible aspects of animal studies, from behavioral and cognitive abilities to imaging and pathology. Such data banks could facilitate multidiscipline studies, save our resources and more importantly save on the number of sacrifized animals. This is an important step towards a better understanding of the pathogenesis of dementia and diabetes, and even starting new trials based on the underlying causes, such as anti-inflammatory medications. Human studies: Using a common vocabulary in definition and categorization of diabetes and dementia, having national / international animal banks and finally a multidimensional approach toward such diseases with multiple and reciprocal presentations and etiologies may lead to optimal human studies and the creation of national/international human data banks. Undoubtedly, we need to provide more sensitive and specific instruments as well as tests to assess diabetes in the first stages and at the same time evaluate a range of cognitive abilities from executive functions, which is more common in vascular cognitive impairment, to memory abilities. In addition, such holistic vision can identify a range of comorbid and confounding disabilities in diabetes, from obesity and vascular diseases to important psychiatric conditions, such as depression. Finally, there is a need for designing multidiscipline studies to assess epidemiological aspects of dementia and diabetes, public health impact and practical clinical trials. Such studies can provide a great chance to detect diseases in pre-symptomatic phases and follow them longitudinally.\n\nMulticenter trials with factorial design, using a combination of lifestyle modifications and medical intervention to diagnose as well as prevent/ treat diabetes and dementia would have paramount of importance in each and every community worldwide.In summary, Stoeckel and colleagues highlight a significant gap of knowledge and consequently appropriate management, definitions, pathogenesis and treatment of dementia and diabetes. A combination of several comorbid and confounding factors and at the same time a possible bi-directional relationship between these common conditions emphasizes an urgent need for a more holistic multidimensional vision to tackle diabetes and dementia together.",
"responses": [
{
"c_id": "2009",
"date": "02 Jun 2016",
"name": "Luke Stoeckel",
"role": "Author Response",
"response": "Referee Reports: 6 May 2016 (MR Azarpazhooh and V Hachinski): We thank the referees for highlighting important strengths of our manuscript: “The authors offer a balanced review regarding the relationship between cognitive decline and insulin resistance. They cover a majority of important studies, providing excellent insights into our current knowledge, and more importantly gaps in definition, pathophysiology, and prevention and treatment of cognitive impairment related to diabetes. One of the strongest parts of the paper is highlighting a probable reciprocal cause and effect relation between diabetes, leading to the cognitive decline, and neurodegenerative lesions in the brain, resulting in central insulin resistance….” Response: We agree with the sentiments expressed by these referees and have expanded the text to further emphasize the major themes identified at the workshop and identified by these referees, and we expanded discussion of these themes to better guide future research in this area (see pgs. 8-9). We request that the list of other research needs be moved to the supplementary materials section."
}
]
}
] | 1
|
https://f1000research.com/articles/5-353
|
https://f1000research.com/articles/5-1087/v1
|
02 Jun 16
|
{
"type": "Review",
"title": "Molecular studies of exercise, skeletal muscle, and ageing",
"authors": [
"James A. Timmons",
"Iain J. Gallagher",
"Iain J. Gallagher"
],
"abstract": "The purpose of an F1000 review is to reflect on the bigger picture, exploring controversies and new concepts as well as providing opinion as to what is limiting progress in a particular field. We reviewed about 200 titles published in 2015 that included reference to ‘skeletal muscle, exercise, and ageing’ with the aim of identifying key articles that help progress our understanding or research capacity while identifying methodological issues which represent, in our opinion, major barriers to progress. Loss of neuromuscular function with chronological age impacts on both health and quality of life. We prioritised articles that studied human skeletal muscle within the context of age or exercise and identified new molecular observations that may explain how muscle responds to exercise or age. An important aspect of this short review is perspective: providing a view on the likely ‘size effect’ of a potential mechanism on physiological capacity or ageing.",
"keywords": [
"human muscle biology",
"ageing",
"excercise",
"skeletal muscle",
"human physiological capacity"
],
"content": "Introduction\n\nIdentification of effective strategies to substantially improve ‘health span’ in humans would revolutionise current approaches to health care. Rather than the current focus on the development of diverse therapies for treating individual age-correlated diseases (cancer, heart disease, and dementia), a strategy that positively impacted on the ageing process would in theory postpone disease. During this extended period of absence from illness, sustained functional capacities (for example, muscle strength and cognitive status) would be key components of ‘healthy ageing’. In turn, these two functional capacities (or approximate correlates) represent key clinical measures that are often used to define the ageing process. There is extensive evidence that exercise capacity (an integrated property of neuromuscular, metabolic and cardiovascular function) is under strong genetic as well as environmental influence. Indeed, key components of exercise capacity (for example, aerobic capacity) vary regardless of the observed range of exercise behaviours1 in older adults.\n\nIn fact, the role of genotype influencing ‘intrinsic’ exercise capacity coupled with the impact of genetics on adaptation to exercise training2 is not the only complication for studying human ageing. The impact of population stratification on the interpretation of cross-sectional analyses exploring the mechanisms for musculoskeletal ageing is typically under-appreciated. For example, over 30% of all cancer-related deaths occur before the age of 64 years (www.cdph.ca.gov), meaning that this subpopulation of individuals can never be included in a cohort of subjects who were at least 70 years old. Notably, the molecular biology of tumour growth and muscle responses to exercise share many common effectors (for example, mechanistic target of rapamycin [mTOR] signalling and pro-angiogenic genes). Furthermore, the ever-popular research strategy3 of comparing the health (or physiology) of sedentary older people (at least 70 years old) with life-long exercisers does not represent an easily interpretable experimental design (for genetic and behavioural reasons). Thus, studying the interaction among individual rates of biological ageing4, exercise behaviour, and molecular factors, including genetics, is clearly non-trivial, and greater caution must be shown when claiming that exercise per se impacts on ageing or health in the elderly. In the present review, we selected about 50 articles from the more than 200 titles published during 2015 that refer to the topic of ‘skeletal muscle, ageing, and exercise’ with the aim of highlighting new understanding or research methods in addition to illustrating technical issues which we feel represent barriers to progress. Studies included further, robust illustration of the highly variable metabolic adaptation noted in response to supervised exercise training5.\n\n\nRecent human studies that address controversies in exercise, ageing, and muscle frailty\n\nThe Kraus laboratory5 demonstrated that improvements in insulin action occurred in fewer than half of overweight subjects undertaking high-volume aerobic or resistance training. Earlier observations found that only the highest training load (something equated to the integral of time/volume) of combined aerobic and resistance training provided a measureable reduction in the group average for indices of glycaemic control6. Interestingly, a correlative analysis this year found that vigorous exercise was most closely associated with modest reductions in all-cause mortality7, and notably combined resistance and aerobic training will result in the exercise sessions including more ‘vigorous’ muscle contractions than those encountered during walking or jogging. Nevertheless, older people in better health (for genetic or other reasons) will be able and potentially may be more willing to undertake vigorous daily physical activity, more so than their unhealthy peers, and this can explain the epidemiological correlation between exercise and health in the elderly. To progress our understanding of the true relationship between ‘exercise’ and health, we need to establish causal mechanisms through randomised clinical trials coupled with global molecular analyses. Such studies will also help evolve a more personalised approach to public health.\n\nUse of the term sarcopenia, which refers to ‘age-associated loss in muscle tissue’, is relatively new8,9 and there has been limited progress in identifying the mechanisms driving sarcopenia in humans. Although both nutrition and exercise habits are postulated to contribute to the prevalence of sarcopenia10, recent studies state that the overall prevalence remains rather low (approximately 5% in patients who are at least 70 years old), but this may reflect the criteria used and the prevalence may be three times greater (but still far from universal). The prevalence of sarcopenia also increases with diseases that impact on mobility11. With respect to inherent defects within the muscle contributing to sarcopenia, the van Loon laboratory found that post-prandial muscle protein synthesis was reduced by approximately 16% in healthy older men versus young men12, independently confirming the anabolic resistance concept first identified by Smith and Atherton13. It is interesting to consider whether the process of sarcopenia is substantially (quantitatively) influenced by insulin resistance. In 2010, it was reported that among 810 Korean subjects, the prevalence of one definition of sarcopenia was approximately 16% in subjects with diabetes versus approximately 7% in the control subjects by 60 years of age14. Others have reported that lean mass is approximately 10% lower in older subjects (about 73 years old) with diabetes and that the rate of loss of computed tomography (CT)-determined thigh cross-sectional area is greater in women, but interestingly not in men, with diabetes15. Whether this is reflective of the molecular features of skeletal muscle in diabetes16 or a consequence of physical activity is less clear (given the lack of direct measures of exercise); the molecular features of sarcopenia, insulin-resistance, and gender need to be compared in detail.\n\nRecently, a meta-analysis by Markofski et al. noted that young and old skeletal muscle does not differ in basal (fasting) protein synthesis rates but that both total and phosphorylated mTOR (a protein complex influencing protein synthesis and breakdown) were elevated in muscle from fasted elderly individuals17. This may reflect a compensatory response, reflecting increased relative muscle loading in daily life (due to reduced muscle strength or conditioning) or may reflect dysregulation of the mTOR complex, which influences muscle mass in a complex and non-linear manner18. Kirby et al. noted that ‘elderly’ mice subject to synergist ablation had lower transcription of ribosomal genes (downstream of mTOR signalling) compared with young mice and that ribosomal gene transcription returned to baseline faster in the old mice19. In model organisms, the mTOR inhibitor rapamycin can extend lifespan. Whilst acute rapamycin administration reduces translation, data this year indicated that chronic administration does not20. More work on the mechanisms underlying rapamycin’s effect on lifespan (mTOR mediated and mTOR independent) as well as the relevance of changes in translation is required.\n\nTherefore, it is still plausible that older people with less sarcopenia start off with greater mobility (or less decline) promoting retention of muscle mass, rather than the development of 'sarcopenia' being the initiating factor. Adding support to this idea, Venturelli et al.21 found that the properties of isolated muscle fibres remained intact in frail older people, while Hart et al.22 found that when physical activity levels were matched (and oxygen delivery capacity was similar), no impact of ageing on plantar flexor oxidative metabolism was noted, suggesting no ‘inherent’ loss of mitochondrial function with age in human muscle. Given the modest prevalence (5 to 15% of those over 70 years old depending on the criteria selected), the contribution of sarcopenia to musculoskeletal frailty (impaired function) in old age remains unquantified. Further, it seems to us that the role for insulin resistance (or obesity) ‘causing’ sarcopenia or frailty has potentially been overstated and only quantitatively modest associations have genuinely been observed. For example, Kim et al.23 found that CT inter-quartile values for thigh cross-sectional area were 1.7 to 2.1 in 50 year olds with normal weight and homeostatic model assessment insulin resistance (HOMA-IR) but were 1.5 to 2.0 in age-matched obese subjects with insulin resistance – hardly a striking difference. Indeed, after studying 482 adults, Loenneke and Loprinzi found that lean body mass was maintained in the face of insulin resistance and inflammation24. Overall, this topic is challenging to address in cross-sectional analyses and using relatively blunt instruments (such as dual-energy X-ray absorptiometry [DXA] or indeed physical activity questionnaires).\n\nOne widely accepted strategy for tackling muscle wasting and weakness is supervised resistance exercise training. Nevertheless, it is still debated whether older people (at least 70 years old) respond in a manner similar to that of younger adults25. Population stratification (as a consequence of ‘premature’ deaths, e.g. cancer) complicates the evaluation of the efficacy of resistance training with respect to human age. This is because up to 25% of healthy adults fail to demonstrate any hypertrophy response to long-term resistance training18,26. Interestingly, a study this year described a lack of hypertrophy response to 6 months of resistance training in a group of otherwise healthy and physically active elderly women (about 68 years old), while on average muscle strength increased by 20%27. The profile of non-responders for gains in function was not reported. The development of diagnostics that could ascertain the proportion of non- or low-responders to resistance training in sarcopenic elderly populations would help clarify the association between molecular potential to ‘grow’ muscle and ability to maintain function with age. Too many human physiology studies undoubtedly report spurious observations owing to small sample sizes and high inter-subject variation, while the use of cross-over designs requires knowledge of appropriate ‘wash-out’ periods. A valid diagnostic could be applied at any age to create a profile of an individual, and this could be used to create better-matched ‘case versus control’ cohorts, each with equal potential for ‘trainability’ or with similar ‘intrinsic’ exercise capacities, when evaluating nutrition or lifestyle interventions.\n\nHowever, it must be pointed out that not all researchers agree on the scale of heterogeneous outcomes obtained from exercise training. The idea that weeks (12 to 24) of supervised resistance training could yield ‘non-responders’ was challenged this year28, using retrospective analysis of about 200 older men and women. The claim that there were no ‘non-responders’ to resistance exercise training was based on the observation that at least one of six (inter-related) laboratory parameters changed in every subject (multiple strength measures, muscle fibre diameter, and lean mass variables). For example, isolated quadriceps muscle function was generally improved in all subjects. However, with an everyday physical test of function (chair-rise time), where accommodation (task learning) is less of an issue, there were non-responders. The study did not formally consider test-retest variation or accommodation or adjust for multiple t tests. Furthermore, although gains in integrated muscle function—reflecting improvements in multiple physiological factors (for example, coordination, muscle mass, and neuromuscular function)—may be more common to most healthy subjects with exercise training, whether this is true for older subjects with pre-existing skeletal problems or disease (for example, diabetes) is less clear25. This returns us to the issue of population stratification, a key challenge for the field to address, ideally using larger-scale longitudinal studies and improved diagnostics of an individual’s potential to respond to standardised physical training.\n\n\nConnecting the dots: acute responses that convey chronic changes in muscle function\n\nThere is an expectation that the molecular and metabolic events that occur during and in the minutes after a single bout of exercise will, over the period of weeks to months, ‘accumulate’ to cause a change in physiological capacity (for example, maximal oxygen consumption [VO2 max] and improved insulin-mediated glucose clearance). Notably, this ‘accumulative’ concept is somewhat contradicted by data from human studies29. Rather than linear accumulating adaptive changes, change may occur through seemingly unrelated secondary molecular responses distinct from acute molecular responses and, critically, be greatly influenced by background genetic and epigenetic factors. There is in fact some pre-existing evidence that muscle molecular responses to acute-on-chronic exercise are rather stochastic29, and this remains one of the most active and challenging topics to study in exercise physiology.\n\nCritically, work in this area has, to date, failed to take advantage of the inherent heterogeneous ability for out-bred mammals to chronically adapt in the face of regular bouts of exercise30,31. In short, acute studies should be carried out in subjects that have been characterised (either by diagnosis or following training + wash-out) for their ability to chronically adapt to the same exercise paradigm. If you accept that the much-ignored issue of population stratification in the older population can result in a different outcome than seen in younger adults (i.e. 75-year-old people are not a random sample of all younger adults), then it is feasible that individuals presenting with musculoskeletal frailty in older age are also those who are less able to mount a muscle hypertrophy response18 or have a more blunted response to nutrition throughout life13 or both. Thus, we will need larger, more sophisticated studies, as well as studies of longer duration, to properly investigate what aspects of musculoskeletal ageing are related to heterogeneous responses to exercise training or nutrition1.\n\nThis year saw the continued search for key molecular ‘targets’ that may govern skeletal muscle size or function32. Most new studies in 2015 focused on consistent molecular responses (group mean differences identified by a paired t test) or used inbred murine models, where variability is understandably much more limited33. It may be more fruitful to stratify acute response against measures of physiological adaptation34. In 2015, two contrasting approaches considered 5′-AMP-activated protein kinase (AMPK) as a candidate transducer for acute-to-chronic adaption. The Wojtaszewski group found that functional gains (endurance capacity) with 4 weeks of training in mice were the same with or without muscle AMPKα1/233 but that acute responses in vascular endothelial growth factor (VEGF) mRNA, a gene involved in vascular adaptation35, was apparently reliant on AMPK. This latter interpretation is probably unreliable, as the AMPK knockout (KO) mouse did 40% less work during the acute bout and, surprisingly, despite also being approximately 50% less physically active, it retained normal insulin and glucose control36. It is plausible that 50% of normal physical activity in ‘cold mice’37 is sufficient to maintain good metabolic health (owing to the very high metabolic turn-over), and careful energy balance studies are needed to establish whether the room-temperature-housed mouse model can ever illuminate mechanisms relevant to human metabolic health.\n\nGenetic and most probably epigenetic factors can influence exercise behaviour and capacity38,39 (for example, altered levels of voluntary wheel running [mice] or aerobic capacity). However, the appearance of specific phenotypes in genetically manipulated mice may or may not reflect the intended genetic manipulation. Indeed, controlling variables beyond the targeted gene when generating transgenic murine models was always deemed complex40 and nearly impossible to achieve with complete certainty, while newer gene editing technology (that is, CRISPR/Cas9) still has off-target concerns41 and detection of individual ‘secondary’ molecular alterations is challenging to spot, as studies are not powered to detect each slight off-target event (but additively they can appear as altered physiology). However, the absence of any effect on chronic adaptation does suggest that AMPK, like PGC1a before it42, is not an essential determinant of physiological responses to exercise. Of course, if hundreds of clinical studies characterise AMPK or PGC1a, then chance associations will still be found (and will be more likely to be published). Nevertheless, we would encourage investigators to move beyond characterising these ‘usual suspects’. In this context, more global (‘unbiased’) research tools provide an advantage, particularly when analysed in a responsible manner43. Such approaches are more likely to uncover more subtle molecular signatures as statistical models (which accumulate small interactions) can be generated, a situation that arguably reflects physiological control.\n\nA recent human study profiling a substantial part of the phosphoproteome identified AMPK-regulated kinases as being the consistent feature of the acute intense cycle exercise in younger males34. This study quantified more than 4300 proteins in human muscle (note that there are about 40,000 RNA molecules detectable in human muscle), identifying many canonical signalling proteins (for example, MAPK, PKA, S6 kinase, and Ca2+ response kinases) and about 600 phosphopeptides that were modified (with mean response statistically consistent). Encouragingly, from our perspective, they found that about 50% (that is, 50% of the shared variance) of the phosphopeptide response to acute exercise was inconsistent across subjects, creating the possibility that studying the highly variable phosphopeptide responses to muscle contraction can help identify factors which contribute to variation in physiological adaptation, if such factors are in operation during acute contraction.\n\nThe caveat to phosphopeptide measurement technology is that at this stage, the overall technical reproducibility of the methodology has not been established. Given that the same researchers have demonstrated that murine muscle AMPK is not essential for physiological adaptation to exercise training33, or fasting glucose and insulin homeostasis for that matter, then the human phosphopeptide responses which are unrelated to AMPK could transpire to be the most interesting to characterise in future human studies. A more detailed discussion of considerations when applying proteomics to the study of muscle and exercise can be found in Padrão et al.44, and this review indicates that the technology is very much a ‘work in progress’. In general, alternative statistical analysis strategies applied to existing data would be beneficial. However, an important challenge is placing the molecular responses within a familiar biological context, as a valid pathway or ontology enrichment (although used) is not possible with ‘proteome’ data because of a lack of knowledge of the detectable background (or ‘universe’) of molecules. Without such information, the regulated list is merely a subsample of the tissue being studied and invariably yields significant enrichment scores versus the ontology database43. Stratification of detected molecules against physiological phenotype seems to us the most productive strategy for human studies in the shorter term.\n\n\nNon-coding RNA that has potential to impact on the biology of exercise, skeletal muscle, and ageing\n\nAmong the most exciting molecular entities contributing to our understanding of the complexity of the genome and physiological regulation are long non-coding RNAs (lncRNAs). Like microRNAs (miRNAs), these transcribed RNAs do not, usually, code for protein. Rather, the transcribed product represents the active molecular effector, regulating the processing of other RNA molecules45 and hence influencing protein expression. lncRNAs are not new to skeletal muscle and exercise; it has been almost a decade since we identified that the PINK1 antisense RNA was regulated by endurance training in humans, altering splicing of PINK1, a metabolic gene linked to Parkinson’s disease45. What has changed is the verified scale of lncRNA expression (www.noncode.org), and this includes human muscle tissue (for example, we routinely detect about 9000 lncRNAs by using Affymetrix HTA 2.0 arrays; Affymetrix, Santa Clara, CA, USA). The Glass laboratory recently described LncMyoD, a gene able to control myoblast proliferation and hence potentially influence muscle regeneration from damage46. They identified more than 1000 intergenic lncRNAs in the murine C2C12 cell line by using RNA sequencing data, many regulated during myotube formation (it would be interesting to know whether the diversity of expressed lncRNAs is greater in primary muscle cells to better match muscle in vivo). Knockdown of LncMyoD hampered myogenesis, reducing the expression of genes expressed in mature muscle cells46, indicating a role for LncMyoD in muscle development and hence potentially in recovery from injury.\n\nOne benefit of research reliant on the detection of RNA molecules is that with a little care47, you can be sure of the identity of the molecule you are quantifying (a situation far removed from antibody-based technologies; see below). However, in the case of lncRNAs, the situation is not nearly as simple as one might hope. The Olson laboratory this year described a lncRNA related to murine exercise performance, but the ‘non-coding’ RNA in this case contained a conserved open reading frame that coded for a micropeptide that played a role in regulating calcium handling in murine ‘fast skeletal muscle’48. So, while one can be sure of the identity of the RNA molecule being measured, emerging evidence this year49 indicates that a lot of the less-well-characterised ‘non-coding’ transcriptome may also contain uncharacterised coding regions, which in turn yield peptides that impact on physiological parameters46,48.\n\nDiscoveries in the field of miRNA biology continue and more families of these short (about 22 nucleotides) molecules have been associated with muscle age or found to be regulated by exercise or both50. In humans, in vivo miRNA abundance does not strongly co-associate with target mRNA levels but rather protein abundance, which, in combination with transcription, regulates muscle phenotype16,51. In our experience, major challenges remain for the miRNA field, as the various technology platforms do not consistently measure each miRNA with the same sensitivity or specificity, and changes in abundance can be rather modest (supporting our model that miRNAs function in a combinatorial manner in vivo in adult human muscle and not individually). Thus, whilst short non-coding RNA molecules still represent a challenge for all high-throughput technologies and inconsistencies between the various manufacturers’ technologies must be expected (as well as lack of conservation of miRNAs between mouse and human), lncRNA molecules are easy to reproducibly measure and represent an exciting new set of molecules regulating muscle biology.\n\n\nProtein detection technologies as a barrier to research progress\n\nProtein is considered by most physiologists to be the main effector of function in the cell, yet, unlike RNA, it is very challenging to quantify or specifically measure. In muscle biology, a particular importance is placed on quantification of protein abundance or enzyme activity reflecting the contractile and metabolic functions of this tissue and historical approaches to studying muscle physiology. This has led to a popular—and, we would argue, mistaken—focus on individual proteins (for example, AMPK) claimed to regulate complex physiological phenotypes and ageing52. It is a simple fact that there are fundamental problems associated with how protein abundance or activity is quantified, and this has greatly impacted on the field of skeletal muscle biology and ageing. Such problems include ambiguity over the veracity of the reagents being used to quantify key protein factors associated with exercise or muscle ageing53. For example, GDF11, a circulating growth and differentiation factor, was claimed to decline with age, ‘controlling’ muscle and vascular ageing54, but was in reality misidentified55. Pharmacologically, it appears that GDF11 can inhibit the potential contribution of skeletal muscle satellite cells to maintenance of adult human skeletal muscle56 and so is plausibly related to human muscle ageing. In this case, both the GDF11 SOMAmer assay (SomaLogic, Boulder, CO, USA) and the GDF11 Abcam Western blot antibody (Abcam, Cambridge, UK) cross-reacted with myostatin55, altering the described age-related changes in GDF11 in mice (while no significant changes were noted in rats or humans with age56).\n\nSo how could such problems be avoided, and why is it a major barrier to progress? In the case of GDF11, experiments using tissue from GDF11 KO mice demonstrated that the ‘protein detection’ technologies were not uniquely detecting GDF11. This simple ‘litmus test’ for assay specificity (arguably the most useful thing one can do with a KO mouse) is not an obligatory prerequisite for publication, but this would be a useful development. Manufacturers could be obliged to provide the negative control tissues and cells to investigators. There is no doubt that lack of specificity of detection technologies yielding positive publications can also drive needless research efforts on the back of mistaken observations. For example, although the technical flaws around various GDF11 detection technologies have been in the public domain since July 2015, and there has been an ongoing and visible debate over the validity of any of the available regents57, a raft of correlative studies using GDF11 antibodies have been subsequently published without any reference to the controversies58. The scale of the problem is substantial; irrespectively of the publication of unambiguous evidence proving that the original lab reagents were faulty. For example, more than 250 articles reporting ‘measures’ of irisin were published between 2014 and 2016 despite multiple articles demonstrating the lack of suitability of available protein detection reagents59–62. Indeed, there is a substantial challenge in ‘pausing’ such activity once the proverbial ‘horse has bolted’. Therefore, failure to remedy high-profile discoveries59 based on flawed protein detection technologies62 is, in our opinion, a major barrier to research progress because follow-on research, motivated by mistaken observations or worse, is a considerable consumer of ‘resource’ (that is, researcher time and reagents) that could be better utilised to advance the field. If retrospective analysis reveals major flaws in published ‘impactful’ research, then editors could act more quickly to flag the problems with the original work, and in more clear-cut cases a ‘no-blame’ retraction should be quickly enforced. Indeed, if strong belief in the original research hypothesis remains, this should provide exceptional motivation to fully replicate the original study using validated reagents. This leaves the finite resources (people and funding) available to study human skeletal muscle and ageing better focused on more legitimate hypotheses or observations. Research into ageing, exercise, and muscle will by its nature be complex and involve multiple molecular measurement technologies, but further effort to improve the specificity of the available tools to quantify protein is, in our opinion, a pressing challenge that the field needs to address.\n\n\nConclusions\n\nThe textbook principles that govern skeletal muscle physiology have until recently been developed on studying small homogenous unrepresentative healthy males63, coupled with an over-reliance on acute exercise studies to study human skeletal muscle phenotype. Although it may be understandable (for example, financially), the lack of longitudinal studies with detailed phenotyping has also meant that our understanding of the impact of ‘ageing’ on human skeletal muscle is based on cross-sectional case-control studies. One general conclusion is that sooner rather than later there must be a shift away from funding small studies to funding larger longitudinal intervention studies capable of delivering more definitive answers. Finally, one might also argue that the greatest scrutiny should always be placed on any discovery that claims that a ‘single’ protein or gene can quantitatively explain ageing or exercise performance; extraordinary claims should always require multiple independent sources of evidence.",
"appendix": "Competing interests\n\n\n\nJAT holds Medical Research Council and European Union funding and has consulted for PepsiCo (Purchase, NY, USA). He is also a shareholder in XRGenomics LTD (Scion House, Stirling). IJG contributes to research on personalised medicine in collaboration with XRGenomics LTD.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nJones DA, McPhee JS, Degens H: Is ageing 'highly individualistic'? J Physiol. 2015; 593(14): 3219. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nTimmons JA: Variability in training-induced skeletal muscle adaptation. J Appl Physiol (1985). 2011; 110(3): 846–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZampieri S, Pietrangelo L, Loefler S, et al.: Lifelong physical exercise delays age-associated skeletal muscle decline. J Gerontol A Biol Sci Med Sci. 2015; 70(2): 163–73. 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}
|
[
{
"id": "14126",
"date": "02 Jun 2016",
"name": "Bret Goodpaster",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14127",
"date": "02 Jun 2016",
"name": "Jamie McPhee",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1087
|
https://f1000research.com/articles/5-1084/v1
|
02 Jun 16
|
{
"type": "Review",
"title": "Dynamics of cell polarity in tissue morphogenesis: a comparative view from Drosophila and Ciona",
"authors": [
"Michael T. Veeman",
"Jocelyn A. McDonald"
],
"abstract": "Tissues in developing embryos exhibit complex and dynamic rearrangements that shape forming organs, limbs, and body axes. Directed migration, mediolateral intercalation, lumen formation, and other rearrangements influence the topology and topography of developing tissues. These collective cell behaviors are distinct phenomena but all involve the fine-grained control of cell polarity. Here we review recent findings in the dynamics of polarized cell behavior in both the Drosophila ovarian border cells and the Ciona notochord. These studies reveal the remarkable reorganization of cell polarity during organ formation and underscore conserved mechanisms of developmental cell polarity including the Par/atypical protein kinase C (aPKC) and planar cell polarity pathways. These two very different model systems demonstrate important commonalities but also key differences in how cell polarity is controlled in tissue morphogenesis. Together, these systems raise important, broader questions on how the developmental control of cell polarity contributes to morphogenesis of diverse tissues across the metazoa.",
"keywords": [
"Border cells",
"notochord",
"collective migration",
"morphogenesis",
"intercalation",
"cell polarity",
"Par polarity complex",
"planar cell polarity"
],
"content": "Introduction\n\nCells in developing tissues exhibit numerous types of polarity, both with respect to the tissue itself and to the axes of the organ or embryo. The fundamentals of cell polarity, especially epithelial polarity, in the early embryo are relatively well understood. Great progress has been made in the decades since the discovery of the Partitioning Defective (Par) genes in Caenorhabditis elegans1,2 and the Planar Cell Polarity (PCP) genes in Drosophila3–6. Throughout animal development, however, various types of cell polarity either have to be maintained or dramatically remodeled for groups of cells and tissues to undergo large scale morphological changes. The mechanisms of how cell polarity is controlled in tissues undergoing complex movements and rearrangements remain under active investigation.\n\nIn this review, we describe the current knowledge of how cell polarity is regulated in two developmental model systems: the Drosophila border cells and the Ciona notochord. Border cells undergo a directed collective migration through an actively developing tissue, whereas the Ciona notochord forms through a series of intricate morphogenetic events, including mediolateral intercalation, cell shape changes, and lumen formation. The Drosophila border cells and Ciona notochord cells both undergo complex, multi-stage tissue morphogenesis processes. Although collective directional migration and mediolateral intercalation are very different, both involve the coordinated behaviors of groups of cells that exhibit multiple, distinct, highly dynamic axes of polarity. While the Par/atypical protein kinase C (aPKC) pathway and the PCP pathway are involved in both border cells and notochord, they vary considerably in their precise roles and relative importance. The seemingly disparate border cell and notochord models highlight important concepts in how different kinds of cell polarity contribute to developing organs and tissues, at both small and large scales.\n\n\nCell polarity in the Drosophila ovarian border cells\n\nMany cell types undergo coordinated multicellular migration in embryogenesis. These so-called migrating “collectives” need to polarize at the group level so that they can reach the correct place at the right time and populate (or produce) tissues and organs with the appropriate orientation. The Drosophila ovarian border cells provide a simple genetic system in which to understand the mechanisms that control collective migration (Figure 1A–C). The Drosophila ovary consists of multiple strings of progressively more mature egg chambers, each of which produces a fertilized embryo7. The egg chamber consists of the oocyte and 15 supporting nurse cells in the center, surrounded by a monolayer of polarized epithelial follicle cells (Figure 1A). In mid-oogenesis, between four and eight follicle cells at the anterior end are induced to form a cluster by a specialized pair of cells called the polar cells. The border cell cluster (including the polar cells) then delaminates from the epithelium. Border cells migrate as a group while navigating their way between the nurse cells to the anterior border of the oocyte, where they stop. The border cell cluster contributes to the formation of the micropyle, which is the sperm-entry pore in the eggshell and is required for fertilization of the oocyte8.\n\n(A–C) Schematic of egg chambers showing the stages of border cell migration during ovarian development. Border cells form at the anterior end of the egg chamber (A), migrate between nurse cells (B), and reach the oocyte at the posterior end (C). For simplicity, individual follicle cell membranes are not shown. (D–G) Close-up view of border cell clusters, and the variety of cell polarities displayed by border cells, at the indicated stages of migration. Polar cells (brown) are always at the center of the cluster. The morphological cell polarities correspond to polarized actin, myosin, lateral, and apical markers, as shown in the key. (D) Pre-migration stage. Border cells exhibit a clear front-rear polarity. Prior to the movement between nurse cells, border cells detach from the basement membrane and delaminate from adjacent epithelial follicle cells. (E,F) Migration stage. Two views of the same cluster are shown: a three-dimensional view (E) and a two-dimensional view through the middle of the cluster (F). At this stage, border cells display inside-outside (E), apical-basal (E) and front-rear (F) polarities. (G) Post-migration stage. Once border cells reach the oocyte, they orient with the apical side touching the oocyte.\n\nBorder cells exhibit and require multiple forms of cell polarity. Border cells initially display a canonical apical-basal polarity because they delaminate from an existing epithelium. For both the follicle cells and the presumptive border cells, the apical side of each cell faces the inside of the egg chamber, contacting the nurse cells and oocyte (Figure 1A). The basal side, on the outer edge of the egg chamber, contacts the basement membrane. The apical side of all border cells thus initially points towards the oocyte and is enriched for the apical complex of Par/aPKC cell polarity proteins: aPKC, Par-3 (called Bazooka, or Baz, in flies), and Par-69,10. The apical edge (front) produces F-actin- and non-muscle myosin II- (myosin-) enriched migratory protrusions11–13. The basolateral polarity proteins Par-1 and Discs large (Dlg) are found at the back, or rear, of the cluster (Figure 1D)14. Visible membrane extensions at the back must retract for border cells to move away from the epithelium.\n\nAs soon as border cells move into the egg chamber, however, they undergo a poorly understood ~90° rotation. The apical side now reorients orthogonal to the direction of migration, as visualized by a strong enrichment of Par-6 and Par-3 proteins at the “top” of the cluster (Figure 1E)9. In a cross-sectional view through the cluster, the polar cells are positioned in the middle, surrounded by border cells radiating out in a “rosette” pattern (Figure 1F)15. The apico-lateral contacts between border cells are enriched for both the Par/aPKC complex proteins and the apical Crumbs/Stardust/PatJ complex9,15,16. The cell adhesion proteins E-cadherin and integrin are located in a complementary pattern along internal basolateral membrane contacts between border cells15,17. Par-3 and Par-6 are required for the characteristic rosette conformation of the cluster during their migration, as well as organization of these other membrane-enriched proteins9,17. At this stage, protrusions now extend from basolateral cell membranes (Figure 1E). As border cells finish their migration, they undergo another rotation, turning so that their apical side again faces the oocyte (Figure 1C,G). This orientation may help establish a continuous epithelial layer that ultimately encloses the oocyte9. Border cells thus retain epithelial apical-basal cell polarity, but this polarity is complex and varies during the course of migration.\n\nA second type of polarity exhibited by border cells is front-rear polarity (Figure 1D,F,G). All migrating cells produce major cellular protrusions at the front that range from thin filopodia to broad lamellipodia18. Front-directed protrusions help cells adhere to migratory substrates, sense directional signals from the environment, and propel the cell forward. Adhesions need to be disassembled in order to retract the cell membrane at the rear19. Directionally migrating collectives produce a characteristic “super-cellular” front to rear orientation across a large number of cells20. In response to a chemotactic guidance signal, border cells extend one or two prominent actin-rich protrusions from the front cell, but not from other cells (Figure 1D–F)21. Multiple growth factors secreted from the oocyte activate receptor tyrosine kinases (RTKs) on border cells22–24. A well-characterized RTK signaling pathway then stimulates the formation of a protrusion, thus setting up front-rear polarity of the border cell group21,25–28.\n\nFinally, border cells exhibit “inside-outside” polarity (Figure 1E)29. The free edge of the cluster establishes external contacts with the migratory substrate, the nurse cells. The rear of each border cell contacts a polar cell at the cluster center, thus producing an inner edge. Inside-outside polarity is reinforced as each border cell stays attached to neighboring cells during their journey. Such orientation appears to prevent migratory protrusions from forming internally between cells of the cluster, similar to other collectives29–31. Adhesion of border cells to the polar cells by E-cadherin is critical for establishing this polarity28.\n\n\nCell polarity in the Ciona notochord\n\nThe Ciona notochord consists of only 40 post-mitotic cells that converge and extend through mediolateral intercalation to form a single file rod (Figure 2A–C). As seen throughout the chordates32, this convergence and extension of the axial mesoderm depends on polarized motility orchestrated by the PCP pathway33. Soon after their final cell division in mid gastrulation, the notochord cells become mediolaterally elongated and aligned with one another, exhibiting mediolaterally biased cell protrusions and mediolaterally biased neighbor exchanges that drive the anterior/posterior (AP) lengthening and mediolateral narrowing of the tissue (Figure 2B,B’). It is important to emphasize that mediolateral intercalation is not a directed migration towards the embryonic midline, but instead an oriented bias in neighbor exchanges with individual cells more likely to move between their medial or lateral neighbors than their anterior or posterior neighbors.\n\n(A–E) Schematics of the entire notochord primordium at successive stages of notochord morphogenesis. Dorsal view with anterior to the left in all cases. (A’–E’) Zoomed in views showing just a few notochord cells with greater detail. Cell nuclei, basement membranes, polarized actin and myosin, and polarized apical and lateral markers are shown as in the key at the bottom of the figure. (A,A’) The notochord initially forms a flat plate of cells that is relatively isodiametric in the AP (anterior-posterior) and ML (mediolateral) axes. No AP or ML polarity has been described at this stage, but there is a slight asymmetry of apical-basal markers on the ventral and dorsal sides (not seen in this dorsal view). (B,B’) The notochord cells become mediolaterally elongated and aligned as they begin to intercalate. The notochord as a whole extends along the AP axis while narrowing across the mediolateral axis. The notochord cells are clearly polarized, with a flat edge contacting the flanking tissues and actin-rich protrusions reaching across to the other side. Cell nuclei are polarized medially. (C,C’) At the end of intercalation, the notochord forms a single-file rod of thin, disk-shaped cells (rectangular in the section shown). A perinotochordal ECM surrounds the notochord. Nuclei are located centrally and squished between the anterior and posterior cell faces. (D,D’) The notochord cells become progressively taller in the AP dimension while narrowing mediolaterally. A new phase of AP polarity becomes evident with nuclei localized to the posterior and myosin enriched to the anterior of each cell. (E,E’) Late in notochord morphogenesis, a distinct circumferential belt of cortical actin forms at the equator of each cell. Extracellular lumen pockets form between each notochord cell. Apical and lateral markers become distinctly polarized between the lumen pocket domain and the remaining donut shaped ring of notochord-notochord cell contacts.\n\nThis mediolateral polarity of tractive cell protrusions is not, however, the only manifestation of planar polarity in the Ciona notochord. As the notochord is intercalating it is also building a perinotochordal extracellular matrix, and the PCP pathway plays a poorly understood role in restricting laminin extracellular matrix (ECM) formation to the perinotochordal surfaces. In embryos carrying a mutation in the PCP pathway component Prickle (Pk), ectopic laminin can be seen between adjacent notochord cells and not just on the outer surfaces of the notochord34. This phenotype is only seen at later stages, indicating that the PCP pathway is involved in the maintenance as opposed to the initial establishment of polarized laminin localization. It is not clear if this control is at the level of laminin secretion, assembly, stability, or otherwise, but a similar relationship between PCP signaling and polarized ECM formation has also been described in Xenopus35.\n\nAfter intercalation, the Ciona notochord cells are in the shape of thin, flat disks (a so-called “stack of coins”; Figure 2C,C’) but they subsequently change shape, becoming narrower across the mediolateral axis and longer along the AP axis (Figure 2D,D’). This change from disk-shaped to drum-shaped accounts for much of the elongation of the tail and is driven by actomyosin-based contractility36,37. Several distinct aspects of cell polarity have been described during this phase of morphogenesis. Cell nuclei become localized to the posterior cell cortex, and this polarity is PCP dependent33. The PCP component Pk becomes polarized to the anterior cell cortex33. Myosin light chain also becomes localized to the anterior cortex in a poorly understood manner that is partially dependent and partially independent of the PCP pathway37. Late in the process, a distinctive circumferential belt of actin becomes evident at the AP equator of the cell36 (Figure 2E,E’), and a recent study shows that the equatorial positioning of this belt involves a balanced interplay between PCP signaling moving the belt towards the anterior and contractility-dependent cortical flows moving it towards the posterior38. The morphogenetic significance of these polarized phenomena is unclear, but it is clear that the intercalated notochord has an AP polarity that is distinct from the mediolaterally oriented planar polarity seen during intercalation.\n\nLater in development, the Ciona notochord forms an inflated lumen, important for its mechanical role acting in compression to cause the larval tail to deflect from side to side instead of collapsing in response to the sequential contraction and relaxation of the left and right tail muscle cells. This central lumen originates from extracellular pockets of material formed between adjacent intercalated notochord cells39 (Figure 2E,E’). These pockets expand and then the notochord cells deintercalate somewhat to allow the pockets to connect and form a continuous lumen running the length of the notochord. This is a topologically confusing rearrangement in 3D that is difficult to depict in 2D. However, the key points are that the lumen pockets are always extracellular and the eventual fusion of multiple pockets results from altered cell-cell contacts, not from membrane fusion events. As such, this does not lead to individual notochord cells becoming donut-shaped.\n\nThis lumen formation process has been extensively studied and involves dramatic changes in cell polarity40–42. The extracellular lumen pockets form only on the anterior and posterior surfaces between adjacent notochord cells and not on the circumferential surfaces between notochord cells and the surrounding muscle, neural tube, and endodermal strand cells. The earliest signs of this polarity come from the localization of the apical markers Par3, Par6 and aPKC to the center of both the anterior and posterior cell surfaces41 (Figure 2E,E’). The basolateral markers DLG, LGL, and Scribble are excluded from this region and are found more peripherally on the anterior and posterior surfaces, as well as the circumferential surfaces that contact the perinotochordal basement membrane. Tight junctions form between these apical and basolateral domains and the extracellular lumen pockets are secreted from the apical region. This leads to the striking implication that the Ciona notochord forms a linear stack of cells that each possesses two distinct apical sides!\n\n\nDynamics of developmental cell polarity: not always epithelial to mesenchymal transitions\n\nCell biologists tend to think of tissues as fitting into the binary categories of epithelium versus mesenchyme. In developing embryos, however, it is clear that these distinctions are somewhat more fluid. There are many morphogenetic processes that involve distinct epithelial to mesenchymal transitions (EMT) or the reverse, mesenchymal to epithelial transitions (MET), but there are also cases where dynamic tissues exhibit both epithelial and mesenchymal characteristics. During classical EMT events, cells lose contact with each other by downregulating apical-basal polarity and E-cadherin-dependent cell-cell adhesion43. Cells then detach from epithelia and the basement membrane to move through tissues. Mesenchymal cells are characterized by the ability to migrate, typically as single cells, and tend to have more elongated “fibroblast-like” shapes44,45. Coincident with becoming mesenchymal, cells often make a switch from E-cadherin to N-cadherin expression43,45. A classic example of a developmental EMT is the departure of the neural crest cells from the neural tube46. Both the Drosophila border cells and the Ciona notochord, however, defy the simple characterization of epithelium versus mesenchyme.\n\nDrosophila border cells, for example, display stretched shapes and extend migratory protrusions, suggesting that they are partially mesenchymal. Similar to cells that undergo EMT, border cells separate from a basement membrane and dissolve contacts with neighboring epithelial follicle cells (Figure 1D)14,21,29. However, border cells never lose contact with neighboring border cells or the pair of polar cells at the center of the cluster. Thus, border cells delaminate and move as a small “epithelial patch”15 (Figure 1E,F). In stark contrast to cells undergoing EMT, apical-basal polarity proteins remain localized to cell contacts between border cells9,17. Moreover, border cells upregulate E-cadherin expression and do not express N-cadherin15,29. Further emphasizing that border cells do not easily fit a stringent definition of either epithelium or mesenchyme, E-cadherin promotes the ability of border cells to migrate upon nurse cells15,28. Loss of E-cadherin in either border cells or nurse cells stops the forward movement of border cells. Dynamic and transient E-cadherin at membrane contacts between border cells and nurse cells therefore provides optimal traction for the cluster.\n\nCiona notochord cells also exhibit aspects of both epithelial and mesenchymal organization. The early notochord primordium forms a flat plate of cells with its dorsal side contacting the neural plate and its ventral side facing the archenteron. These two tissue surfaces differ in both the onset and progression of cell shape changes47 and show accumulation of the apical marker aPKC ventrally and the basal marker laminin on the dorsal side48. Despite these clear signs of epithelial organization, cells in this tissue are nonetheless able to undergo repeated intercalation events and change nearly all of their neighbor-neighbor relationships. After intercalation, it is not until quite late in tail extension that they again show clear signs of epithelial polarity, as they adopt their unusual biapical organization in preparation for lumen formation41.\n\n\nDiffering requirements for the PCP pathway\n\nBorder cells essentially move in a planar direction because the apical side relocates to the top of the cluster during migration. Three PCP pathway members, Frizzled (Fz), Dishevelled (Dsh), and Strabismus (Stbm; also known as Van Gogh), are each highly expressed in the central polar cells prior to migration, in addition to low levels in border cells49. Once the cluster moves into the egg chamber, Fz localizes to the leading edge in association with F-actin rich protrusions, whereas Dsh and Stbm are found throughout the cluster49. Given the PCP expression in border cells, it is surprising that loss of any one of these genes only mildly perturbs border cell migration. Knock down of fz, dsh, or stbm in border cells causes slight delay in cluster migration, although most clusters make it to the oocyte by the correct stage of development49. Mosaic clonal analyses with PCP mutants indicate that the PCP genes help establish or maintain a lead cell identity, as well as communication between polar cells and border cells49. In addition, F-actin enrichment in border cells via the RhoA GTPase pathway requires the function of the PCP genes49. PCP signaling may therefore contribute both to inside-outside polarity and to front-rear polarity during border cell migration. The role for PCP pathway components is minor, however, suggesting functional redundancy with other mechanisms.\n\nThis modest role for PCP signaling in border cell migration stands in stark contrast to the diverse and essential roles of the PCP pathway in Ciona notochord morphogenesis. A mutation in the PCP pathway component Pk eliminates the mediolateral bias in the orientation of tractive cell protrusions and prevents the notochord plate from intercalating into a single file rod of cells33. These Pk mutant embryos also show defects in perinotochordal/intranotochordal polarity, as seen by the ectopic localization of laminin to some surfaces between adjacent notochord cells34. Despite the major defect in convergent extension, some cells in the posterior notochord do typically intercalate but exhibit a later defect in AP polarity, as shown by nuclei that fail to be positioned to the posterior of each cell33. While it is not clear to what degree these three different aspects of cell polarity depend on one another, it is clear that the PCP pathway plays major roles in mediolateral polarity, inside versus outside polarity and AP polarity in the Ciona notochord.\n\nThe role of the PCP pathway in the convergence and extension of chordate axial mesoderm appears to be very broadly conserved32. Interestingly, this is true even in species that employ very different cellular mechanisms of convergence and extension. In zebrafish, for example, convergence and extension is dominated by directional flows of independently migrating cells as opposed to mediolateral intercalation per se, but the PCP/non-canonical Wnt pathway is nonetheless essential for these movements (reviewed by 50).\n\nDespite this conserved role in axial elongation in chordates, it is also interesting to note that the PCP pathway does not appear to be a main driver of the corresponding movement of germband elongation in the Drosophila embryo, which is also a tissue rearrangement defined by mediolaterally biased cell-cell neighbor exchanges51–53. A potential explanation for this discrepancy lies in the arguably distinct cellular mechanisms of intercalation. Drosophila germband elongation is thought to depend largely on neighbor exchanges driven by polarized contractility of mediolaterally oriented cell-cell contacts54,55, whereas research into chordate mediolateral intercalation has emphasized the role of mediolaterally polarized cell protrusion interdigitating between adjacent cells. However, recent research has shown an important potential role for polarized contractility in Xenopus mediolateral intercalation56, and it is possible that these two modes of intercalation may be more alike than previously acknowledged, despite the differing importance of PCP signaling.\n\n\nApical-basal polarity and morphogenesis\n\nThe apical epithelial Par/aPKC pathway plays important roles in border cell cluster organization and migration. Loss of Par-3 or Par-6, or an upstream activator c-Jun N-terminal kinase (JNK), disrupts the organization of membrane-enriched proteins, such as E-cadherin and integrin, between border cells9,17. Individual mutant border cells then partially pull away from the main body of the cluster and extend ectopic stable protrusions. Border cell clusters mutant for Par-3, Par-6 or JNK also do not complete their migration to the oocyte9,14,17. In many directionally migrating cells, apical polarity complexes, including Par/aPKC, promote formation of the leading edge57–59. Although front-directed protrusions are critical for border cell migration, the role for Par/aPKC was unclear. A recent study connects polarized protrusion formation with apical-basal polarity at the cluster level16. RTK-dependent guidance signaling triggers F-actin-rich protrusions at the cluster front through activation of Rac GTPase26. Loss of Pak3 (p21-activated kinase 3) causes border cells to extend ectopic unstable protrusions and stalls their migration16. Importantly, Pak3 functions downstream of the RTKs, but upstream of JNK, to control the proper localization of apical-basal polarity proteins within the cluster16. The Par/aPKC apical complex thus promotes cohesion of the border cell collective and facilitates directional migration, likely through Pak3-dependent polarized protrusion extension.\n\nThe basolateral protein Par-1 has a slightly different role in border cells than the Par/aPKC apical complex. Par-1 promotes the complete separation of border cells from the adjacent follicle cell epithelium13,14. In epithelia, Par-1 at the basolateral side phosphorylates several conserved Ser/Thr residues, which prevents apical Par-3 from relocalizing to the basolateral membrane60,61. Similarly, Par-1 restricts localization of apical Par-3 at membrane contacts between border cells and adjacent follicle cells, leading to downregulation of E-cadherin between the two cell types14. The mechanism by which loss of Par-3 leads to loss of cell-cell adhesion is still unclear. Polarized actomyosin contraction of the cluster rear also contributes to the delamination process13. Par-1 promotes localized myosin activity to the rear of the cluster prior to its movement out of the anterior epithelium. Par-1 also plays a poorly understood role in promoting the formation of front-directed protrusions, which is independent of its role in regulating Par-3 localization14. Par-1 thus contributes to front-rear polarity, at least during delamination of border cells.\n\nWhile many details remain to be resolved, it is clear that canonical apical/basal polarity pathways play key roles in several aspects of border cell migration. This is quite different than in the Ciona notochord, where a major role for the Par/aPKC pathway has only been demonstrated quite late in tail elongation as the intercalated and elongated cells develop their unique biapical configuration in preparation for lumen formation41. A modest dorsoventral asymmetry in aPKC has been described prior to intercalation in the notochord plate, but a strong asymmetry in Par/aPKC components has not been observed during intercalation or in the earlier stages of the post-intercalation notochord cell shape changes. Similarly, perturbations of the Par/aPKC pathway have been shown to have lumen secretion phenotypes but do not affect these earlier processes41. It is possible that the Par/aPKC pathway has roles in intercalation that remain to be discovered, but it does not appear to be a major driver. One possible interpretation is that the intercalating notochord cells have a basal, ECM-facing, perinotochordal side but that the intranotochordal surfaces are not yet apical in any meaningful sense, instead representing a naïve condition.\n\nA potential explanation for the very different roles of the Par/aPKC pathway in border cell migration versus notochord morphogenesis is that the border cells delaminate from an indisputably epithelial cell layer, whereas notochord morphogenesis occurs in the early embryo as the first basement membranes and the first axes of cell polarity are being established. It is clear that there are major “chicken and egg” questions about cause and effect with respect to the origins of polarized ECMs and polarized cell morphologies in early embryos.\n\n\nKnowing where you are: inside-outside polarity\n\nThe free edges of migrating collectives make contacts with the ECM of other cells. Cells at the periphery of a group thus exhibit a distinct inside-outside polarity that internal cells do not. For border cells, the internal edge contacts a central polar cell, whereas the external free edge contacts a nurse cell. The adherens junction protein E-cadherin plays a pivotal role in defining this polarity. E-cadherin is highly enriched at intra-cluster contacts between border cells and polar cells9,15. Loss of E-cadherin only in polar cells causes border cells to completely detach from the cluster28. In contrast, depletion of E-cadherin only in border cells does not disrupt their adhesion to each other15,28. Thus, by keeping border cells in contact with the polar cells, E-cadherin establishes an inside-out polarity for each border cell. At the outer edge of the cluster, low levels of E-cadherin facilitate dynamic adhesion of border cells to the nurse cells15,28.\n\nActomyosin enrichment further defines the outer edge of the cluster. F-actin and activated myosin are normally restricted to the external edge of the cluster, with low levels found at internal border cell membranes (Figure 1F)13,27,30. Other migrating collectives display similarly polarized myosin at outer edges62–64. Increased activation of myosin at internal cell-cell contacts disrupts cohesion of collectives63. Such polarized actomyosin activity mechanically joins cells together to promote group movement64,65. In border cells, the Hippo pathway specifically regulates the functions of actin-regulatory proteins to promote F-actin polymerization at the outer edge30. Hippo prevents ectopic enrichment of F-actin and activated myosin at contacts between border cells. Actomyosin tension on the outer edge of the cluster is important for cluster motility27,30.\n\nCiona notochord cells also exhibit an inside-out polarity, with the perinotochordal basement membrane only forming on the outer surfaces touching the flanking muscle, neural tube, and endodermal strand cells, and not on the inner surfaces touching other notochord cells. This polarity is at least partially PCP dependent34, but is distinguishable from the polarity that drives intercalation. In this case, perinotochordal basement membrane forms on all of the notochord-to-not notochord cell surfaces, but tractive protrusions on the notochord-to-notochord cell surfaces are heavily mediolaterally-biased33,47.\n\nAs intercalation proceeds, a distinctive ventral groove forms along the midline of the notochord primordium47. It is not clear if this represents a dorsoventral difference in the speed of intercalation or a contraction of ventral cell surfaces, but it arguably transforms the dorsal surface of the notochord plate into the outer surface of the intercalated notochord. Overexpression of a dominant negative ephrin disrupts the apicobasal polarity of the notochord plate and blocks intercalation, suggesting important but cryptic functional relationships between these apicobasal, inside-out and mediolateral manifestations of polarity48.\n\n\nCytoskeletal readouts of polarity\n\nDynamic changes in cell polarity are ultimately manifested as polarized cell behaviors, many of which are driven by the cytoskeleton. A key distinction here is in the ability of the actomyosin cytoskeleton to both pull and push depending on the local architecture of the actomyosin network66. While not the focus of this review, many important invagination and folding events in developing embryos are driven by apical constriction, in which polarized contractility plays the central role and polarized protrusions are not involved67. More frequently, however, the same cell will demonstrate both protrusive and contractile behaviors. This is particularly true for migrating cells or cell collectives in which the leading edge has polarized lamellipodia and filopodia, whereas localized contractility ensures the retraction of the trailing edge. The interplay between protrusive and contractile behaviors is difficult to study because the relevant structures are small and extremely dynamic in time and space.\n\nThe convergence and extension of the Ciona notochord involves at least two distinct mechanisms influencing protrusive cell behaviors. The PCP pathway plays the major role in restricting the cell protrusions driving intercalation to medial and lateral surfaces33, though it remains unclear what sort of broader cues are used to align this planar polarity with the axes of the embryo. As cell protrusions reach across to contact the flanking cells on the opposite side of the embryo, these protrusive behaviors quickly cease and the cell flattens out against the adjacent muscle, neural, or endodermal strand cell. This boundary capture requires an intact perinotochordal ECM, as it is badly perturbed in a mutant for a notochord-expressed laminin alpha3/4/534. In this mutant, the notochord cells exhibit persistent motility and intercalate inappropriately between many of the cells surrounding the notochord.\n\nThe subsequent shape changes in the notochord are driven by actomyosin contractility, but it remains unclear to what extent polarized contractility is important. Myosin becomes enriched to the anterior early in this process37, and a distinctive circumferential belt of actin forms somewhat later36; however, it is experimentally difficult to disentangle the relative importance of contractility on these different cell surfaces.\n\nIn Drosophila border cells, the formation of an F-actin-rich protrusion in the lead or front cell is a major manifestation of cluster-wide front-rear polarity11,12. Regulation of polarized border cell protrusions was a mystery prior to the discovery of ex vivo culture conditions that supported egg chamber development21,25. Live time-lapse imaging of migrating border cells reveals the remarkable dynamics of these protrusions. Border cells can extend and retract a long lamellipodia-like protrusion multiple times before moving out of the epithelium. Retraction of this protrusion requires actomyosin contraction11. Once the cluster moves into the egg chamber, only the front border cell produces a stable protrusion. Major protrusions are actively suppressed in the rest of the border cells.\n\nA strikingly complex RTK pathway sets up which border cell will produce a migratory protrusion. Border cells are guided to the oocyte by RTK signaling22–24. Multiple growth factors are secreted by the oocyte to activate two RTK receptors, platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) related receptor (PVR) and epidermal growth factor receptor (EGFR), on border cells. The lead border cell is thought to receive the highest level of RTK activation25. PVR and EGFR then induce Rac GTPase activity at the front of the cluster26. This in turn leads to accumulation of F-actin in the lead cell and extension of a protrusion22,26. Ectopic Rac activation in any cell during live migration is sufficient for that cell to produce a stable protrusion and steer the group in a new direction26. Further, the endocytic Rab11 protein promotes cluster-wide cell-cell communication to suppress protrusions in non-leading border cells27. E-cadherin-based tension at the front of the cluster reinforces all of these signals, such that only the front cell forms a protrusion28. The end result is that the border cell cluster moves to the oocyte in a directional manner22,23,26.\n\nFinally, it is worth noting some striking similarities and differences between border cell collective migration and a distinctive morphogenetic process that occurs in the developing Drosophila eye. The fly eye is made up of repeating units of ommatidia, each of which consists of eight photoreceptor cells surrounded by twelve support cells5. The cells in each ommatidium undergo a collective rotation in which they move together as a group to rotate 90°. The direction of rotation is flipped on opposite sides of the eye imaginal disc midline. Similar to border cell migration, ommatidial rotation is also regulated by RTK signaling, with EGFR being required to complete the full 90° rotation68–70. However, PCP signaling is the predominant regulator of this polarized collective behavior4,5, with mutations in core PCP genes such as fz causing random rotation of ommatidia71. In border cells, the roles of these two pathways are quite different. Two RTKs, EGFR and PVR, guide border cells to the oocyte and maintain their motility by helping produce front-rear polarity, whereas PCP signaling is less important21–23,25,26,49.\n\nBoth border cell migration and ommatidial rotation also involve E-cadherin as an important effector molecule. The function of E-cadherin in border cells is complex; it promotes migration of border cells upon nurse cells, keeps border cells attached to the central polar cells, and confers cluster-wide polarized tension15,28. In ommatidia, loss of E-cadherin results in incomplete rotation72. Interestingly, another classical cadherin, N-cadherin, limits rotation, so that ommatidia do not over-rotate. Cadherin regulation ties into the PCP pathway through a MAP kinase family member called Nemo72,73. Nemo physically binds to the PCP proteins Stbm and Pk, and regulates the activity of E-cadherin complexes during rotation through the cadherin binding partner ß-catenin73. Rho family GTPases, myosin, and diverse F-actin regulatory proteins also function as downstream effectors in the movement of both cell types11,29,49,74–76.\n\n\nQuestions for the future\n\nIn comparing Drosophila border cells and the Ciona notochord, several themes emerge that raise further questions. Many developing tissues can simultaneously, or successively, display concurrent types of morphological polarization that include apical-basal, mediolateral, AP, and inside-outside cell polarity. To what extent are these different types of polarity contingent on one another? How often are they truly orthogonal? Dynamic and rapid transitions from one type of cell polarity to another can occur at different stages of development. What are the “switches” between successive phases of cell polarity? And to what extent do they depend on changes in cell-cell communication and gene regulatory networks versus biophysical changes in cell and tissue architecture?\n\nMany tissues exhibit intermediate states of organization that are neither strictly epithelial nor strictly mesenchymal. What is the best way to think about developing tissues that show signs of both epithelial and mesenchymal organization? One likely answer is that intermediate states are important for the ability of cells to move as groups, and to sculpt and remodel organs during embryonic development. Why some developmental processes, but not others, rely on such a mixture of epithelial and mesenchymal properties is currently unclear.\n\nAlthough the Par/aPKC and PCP pathways are known to organize diverse aspects of polarized cell morphology and behavior, the precise roles and relative importance of these mechanisms vary dramatically in different contexts. Despite an increasingly detailed understanding of the molecular intricacies of the Par/aPKC and PCP pathways, major questions remain as to the mechanisms underlying the temporal and spatial control of the dynamic transitions between successive aspects of cell polarity during development of specific tissues and organs. Dynamic changes in cell and tissue polarity are also central to tumor invasion and metastasis and, while it is well established that polarity proteins and programs are disrupted in many cancer cells77,78, we generally lack a rich and mechanistic understanding of how this dysregulation occurs.\n\nAnswering these and other questions raised by the border cell and notochord systems will help our understanding of how cell polarity contributes to the dynamic formation and remodeling of developing tissues. A better understanding of how cell polarity is established, maintained and transformed will provide a foundation for understanding how stem cells can be used therapeutically to build new organs, and how cancer cells co-opt these programs during tumor progression.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work was supported in part by National Science Foundation grants IOS-1456555 to MTV and IOS-1456053 to JAM, and by National Institutes of Health grants 1R01HD085909, P20GM103638 (CMADP COBRE), P20GM103418 (Kansas INBRE) to MTV and R21CA198254 to JAM.\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\nKemphues KJ, Priess JR, Morton DG, et al.: Identification of genes required for cytoplasmic localization in early C. elegans embryos. Cell. 1988; 52(3): 311–20. PubMed Abstract | Publisher Full Text\n\nGoldstein B, Macara IG: The PAR proteins: fundamental players in animal cell polarization. Dev Cell. 2007; 13(5): 609–22. 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PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDenker E, Bocina I, Jiang D: Tubulogenesis in a simple cell cord requires the formation of bi-apical cells through two discrete Par domains. Development. 2013; 140(14): 2985–96. PubMed Abstract | Publisher Full Text\n\nDeng W, Nies F, Feuer A, et al.: Anion translocation through an Slc26 transporter mediates lumen expansion during tubulogenesis. Proc Natl Acad Sci U S A. 2013; 110(37): 14972–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLim J, Thiery JP: Epithelial-mesenchymal transitions: insights from development. Development. 2012; 139(19): 3471–86. PubMed Abstract | Publisher Full Text\n\nFriedl P, Wolf K: Plasticity of cell migration: a multiscale tuning model. J Cell Biol. 2010; 188(1): 11–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYe X, Weinberg RA: Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression. Trends Cell Biol. 2015; 25(11): 675–86. 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PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "13419",
"date": "02 Jun 2016",
"name": "Marek Mlodzik",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13420",
"date": "02 Jun 2016",
"name": "Di 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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1084
|
https://f1000research.com/articles/5-1061/v1
|
01 Jun 16
|
{
"type": "Method Article",
"title": "Making dimers of oligomeric membrane proteins using copper-free click chemistry",
"authors": [
"Wang Dong",
"James N. Sturgis",
"Wang Dong"
],
"abstract": "Here we describe the development of a protocol to make small oligomers, dimers and trimers, from highly oligomeric membrane proteins. The proteins that we used are the light harvesting 2 proteins and core complexes from photosynthetic bacteria, which contain respectively 16 and 56 individual polypeptides. Creating specific dimers between such multimeric protein poses several problems. We propose a protocol based on asymmetric lysine localization, thanks to the positive inside rule, and copper-free click chemistry. With this method we are able to produce specific dimeric complexes in detergent solution of possible biological relevance.",
"keywords": [
"photosynthesis",
"light harvesting",
"click chemistry",
"Rhodobacter sphaeroides",
"Roseobacter denitrificnas",
"Phaeosprillum rubrum",
"core complex"
],
"content": "Introduction\n\nThe light harvesting apparatus of photosynthetic bacteria, and indeed of all photosynthetic organisms, is organized in a large array of complex structure. This array is designed to absorb light energy and efficiently funnel it to available reaction centers1. In most purple bacteria the light collection array is comprised of two proteins: the reaction center containing core complex (CC), and the peripheral light harvesting complex (LH2). In Figure 1A an AFM image of a fragment of photosynthetic membrane from Rhodospirillum photometricum is shown, immediately apparent is the extensive array of proteins. The visible proteins are of two types: smaller rings composed of LH2, a molecular model of which is shown in panel B, and larger rings composed of CC, a molecular model of which is shown in panel C. Analysis of the organization of these proteins2 has shown that this organization can be described as a mixture of hexagonal packed arrays of the nonameric LH2, and a more randomly organized LH2 CC mixture.\n\nA, AFM image of a fragment of photosynthetic membrane from Rhodospirillum photometricum showing the organization of LH2 (small rings) and CC (larger rings) in the membrane. B, Molecular model of LH2 viewed perpendicular to the membrane surface, the α polypeptides in green and β in red. C, Similar view of a molecular model of the CC, again the α subunits are in green and the β in red, the reaction center C,L,M and H subunits are in magenta, orange, yellow and cyan respectively.\n\nThe individual protein rings shown in Figure 1B & 1C are each oligomeric. The LH2 ring (Figure 1B) is made of a circular array, of usually 9, subunits each composed of two polypeptides α and β, shown in green and red respectively, which bind four pigment molecules, 3 bacteriochlorophyll (BChl) a and one carotenoid molecule3,4. In the case of Phaeospirillum (Phsp.) molischianum the ring is composed of 8 subunits5. The CC is slightly more complex, and also variable, in architecture. In the center is a reaction center component, typically made of the subunits H, M and L with a bound tetraheme cytochrome subunit C, these are shown in Figure 1C colored cyan, yellow, orange and magenta respectively. This is surrounded by the light harvesting complex oligomer of 16 α and β subunits. These subunits are similar to those of LH2 but only bind 2 BChl a molecules6. There is considerable inter-species variability in the precise organization of the CC, with variants having less than 16 subunits, and additional subunits that do not bind pigment or complexes forming S-shaped dimers7.\n\nIn order to better understand energy flow within the light harvesting array8 it would be useful to study isolated parts of the array, larger than a single complex but smaller than the whole array, and with defined architecture.\n\nThis seemingly simple task has proved surprisingly complex. The main difficulties are the large size of the array in situ, the oligomeric structure of the pigment-protein complexes and the need to use detergents as membrane proteins are insoluble in standard biochemical buffers. Initial experiments9,10 with chemical cross-linking in membranes gave very complex mixtures of intra-molecularly linked proteins, inter-molecularly linked homodimers and heterodimers with altered purification properties rendering the objective of reasonably homogeneous samples of defined architecture unattainable.\n\nHere we develop a protocol that allows us to form small relatively homogeneous oligomers (dimers and trimers) in a controlled manner. We show that the yields for the different steps are reasonable and that the end product is as expected.\n\n\nMethods\n\nThe various pigment protein complexes were purified by standard methods as described previously11. Briefly photosynthetic membranes were solubilized with dodecyl-maltoside and pigment protein complexes isolated by sucrose density gradient centrifugation followed by anion exchange chromatography on a resource-Q column and gel filtration on a superose-6 column. The purity of the various complexes was evaluated by absorption spectroscopy (Shimadzu UV1800 spectrophotometer), measuring the ratio of the absorption peaks at 280 nm and 370 nm. This ratio varies somewhat depending on the source, but absorption ratios of 0.3 and 0.65 are typical for purified LH2 and CC respectively. Purified LH2 and CC proteins from Phsp. molischianum, Rhodobacter (Rb.) sphaeroides and Roseobacter (Rsb.) denitrificans were prepared in 20 mM sodium phosphate buffer (pH 7.2) containing 0.05% dodecyl maltoside. For each purified complex extinction coefficients were calculated from a knowledge of the stoichiometry, thanks to the known structures of the complexes, and BChl a extraction from purified complexes as described previously12. Briefly, the UV-visible absorption spectrum was measured, the BChla concentration of the sample was determined from the absorption of an acetone methanol (7:2) extract using the extinction coefficient of ϵ770nm 76 mM–1cm–113.\n\nThe stoichiometry of the complex was calculated as 4 BChl per reaction center plus 2 BChl per LH1 type αβ pair for core complexes and 3 BChl per LH2 type αβ pair for LH2 complexes. The complex extinction coefficient was then calculated as ϵ(cm–1mM–1) = Absorbance ͙ stoichiometry/(1cm͙ [BChl]), see Dataset 1.\n\nSuccinimidyl esters, (5/6-carboxyfluorescein succinimidyl ester and succinimidyl-2-(biotinamido)ethyl-1,3- dithiopropionate), were purchased from Thermo Fisher Scientific (Waltham, USA) and dissolved in dry DMSO from Acros (Geel, Belgium) prior to use. Primary amine labeling was carried out at 4°C for 1 hour in 20 mM Na Phosphate buffer pH 7.2 containing 0.05% dodecyl maltoside.\n\nMaleimides, dibenzylcyclooctyne-PEG4-maleimide and azido-PEG3-maleimide, were purchased from Jena Bioscience and dissolved in dry DMSO prior use. Sulfhydryl labeling was carried out at 25°C for 2 hours in 20 mM Na Phosphate buffer pH 7.2 containing 0.05% dodecyl maltoside. Coupling by copper-free click chemistry was performed in the same buffer for 10 hours at 4°C.\n\nAfter reaction with 5/6-carboxyfluoresceine succinimidyl ester and the maleimides the labeled protein was separated from unreacted label using spin columns (Micro Biospin TM6 columns, Bio-Rad (Hercules,USA)), according to the manufacturer’s instructions.\n\nReaction products after coupling were analyzed by HPLC. 20–40 μl samples were injected and separated on a Agilent technologies (Santa Clara, CA) 1260 infinity chromatography system equiped with a Biosep-SEC-s4000 analytical column (300mmx4.60mm) eluted with 20 mM Na Phosphate buffer pH 7.2 containing 0.05% dodecyl maltoside at a flow rate of 0.5 ml/min and followed by absorption at 280 nm. Absorption spectra of peaks were obtained from the integrated spectral detector (Agilent technologies G1315D diode array detector).\n\nMolecular models of proteins were prepared based on homology to proteins of known structure. In the case of LH2 the structures of the proteins from Rhodopseudomonas (Rps.) acidophila14, and Phsp. molischianum5 were used as templates. For core complexes the recent structure of the protein from Allochromatium (Ac.) tepidum15 was used as a template.\n\nModel structures were visualized and diagrams prepared with Pymol molecular graphics program, version 1.816.\n\n\nResults\n\nThe general approach that we used is shown in Figure 2. In the first step the most reactive lysines in the complex are reacted with a succinimidyl-ester at a very low degree of labeling to ensure on average less than 1 reacted lysine per complex. This low degree of labeling is essential to ensure that during cross-linking the number of higher order oligomers formed is minimal. The choice of an amine directed reagent was governed by the known structure of the complexes where, thanks to the positive inside rule17, a number of lysines predicted to be reactive are routinely found in the exposed cytoplasmic parts of the light harvesting ring. Indeed examination of the structures of LH2 and CC show that there is a ring of exposed lysines close to the cytoplasmic surface of the membrane.\n\nComplexes appropriate for copper-free click chemistry, labeled with DBCO- or azido- groups, were prepared in a two step procedure that allows affinity purification of a biotinylated intermediate.\n\nSingly labeled proteins prepared by partial reaction of lysines were then purified, thanks to the use of a biotin containing reagent, by affinity on streptavidin beads, and eluted by reduction of the dithiol linkage in the reagent. The resulting proteins contain a unique reactive thiol, the proteins used do not have exposed cysteines. So in the next step of the protocol the thiols are reacted with maleimides appropriate for click chemistry. Allowing the formation of self-non reactive singly labeled proteins that can react with each other, an azido-labeled protein with an alkyne-labeled one.\n\nCurves are shown for two different purified complexes: LH2 of Phsp. molischianum (green), and CC of Phsp. molischianum (blue) (Dataset 2).\n\nCritical for the general protocol outlined above (Figure 2) is the possibility of obtaining protein with a controlled low level of labeling on lysine residues. To assess this and verify that even under these somewhat non standard conditions we could obtain reproducible labeling we used carboxyfluoresceine succinimidyl ester to follow the degree of labeling. In Figure 3 we show the degree of labeling observed by absorption spectroscopy using the most red Bchl a absorption band, with the extinction coefficients calculated as described above, and the fluorescine absorption at 494 nm, and the published extinction coefficient of 70 mM–1cm–118. This graph shows that the degree of labeling was linear in the amount of succinimidyl-ester added without any noticeable threshold for labeling. The slopes of the relationship varied between proteins, presumably reflecting differences in the reactivity of the targeted lysines. In Figure 3 we show curves for CC (blue) and LH2 (green) of Phsp. molischianum, the slopes of the labeling relationship for all the different complexes we examined vary over a relatively narrow range of 0.98 to 0.81.\n\nAs the first step was designed to give a very low level of labeling, limited by the amount of succinimidyl ester, it was necessary to separate the labeled protein from unlabeled protein. To achieve this efficiently we chose to use succinimidyl-2-(biotinamido)ethyl-1,3- dithiopropionate, this reagent allows us to strongly and specifically bind labeled protein to streptavidin beads, and elute the protein either with biotin or by reducing the disulfide bond.\n\nTests showed that, as expected, the binding was specific and typical yields with unlabeled protein, containing no bound biotin groups, was less than about 0.1% (the estimated detection limit). In contrast estimated yields of biotin containing proteins were greater than 95%.\n\nWe chose to use disulfide reduction to elute the proteins from the streptavidin beads, this approach gave better yields for the reacted proteins. Two different reducing agents were tested: tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT). Both proved able to release bound reacted proteins. However some proteins, notably several core complexes, proved rather sensitive to TCEP reduction losing their colored cofactors. We therefore decided to use DTT as the reductant, adding DTT to a final concentration of 50mM and allowing reduction to proceed for 4 hours at 25°C. The eluted protein was separated from excess reductant by gel filtration on a spincolumn.\n\nThe selectivity and specificity of this purification method ensures that the majority of the eluted protein, even at very low labeling levels will have one or more reacted lysines. For example, if a degree of labeling of 10% is targeted for a nonameric protein the probability of labeling on of the 9 equivalent most reactive lysines is 1.1% from this, assuming independent labeling of the different groups, we expect the reaction mixture to contain 90.4% unlabeled protein, 9.1% singly labeled protein and 0.5% multiply labeled protein. After purification, given the selectivity and specificity, the mixture is expected to contain less than 1% unlabeled protein and less than 5% doubly labeled protein and 94.6% singly labeled protein.\n\nInitial attempts to cross-link complexes using copper catalyzed click chemistry indicated that the colored cofactors were rather sensitive to monovalent Cu and lost their color under typical reaction conditions. Again this was particularly noticeable for CC and the B800 absorption band of LH2 complexes. In view of this we decided to use a copper-free click chemistry protocol for cross linking based on the strained cyclo-octyne ring19. Aliquots of the proteins to cross-link were reacted with a 50 fold excess of dibenzylcyclooctyne-PEG4-maleimide or azido-PEG3-maleimide at room temperature for 1.5 hours. Unreacted reagent was removed by passage of the reaction mix over a spin column.\n\nImmediately after preparation of the azide and cyclooctane derivatives they were mixed in a 1:1 molar ratio and allowed to react at 4°C over night. After the reaction was complete the sample was analyzed by HPLC.\n\nA, HPLC trace showing absorption at 280 nm of the reaction mixture separated on an analytical Biosep-SEC-s4000, in 20 mM Na phosphate buffer pH 7.2, containing 0.05% Dodecyl maltoside, at a flow rate of 0.5ml/min (Dataset 3). B, Absorption spectra of the major peaks in the HPLC trace: 5.84 min (blue), 6.13 min (magenta), 6.60 min (red) (Dataset 4).\n\nReaction products were analyzed by HPLC on a size exclusion column. Typically, as shown in Figure 4A, several peaks could be observed, the first peak eluting at 5.84 min is specific to samples in which the alkyne and azide were present and corresponds to the cross-linked product. This is followed by peaks, at 6.13 and 6.6 minutes, corresponding to the unreacted CC and LH2 proteins respectively. Several smaller peaks can be observed in the later part of the chromatogram.\n\nThe identity of the different peaks was confirmed by absorption spectroscopy as can be seen in Figure 4B. The second and third peaks show the expected absorption of CC and LH2 respectively, while the first peak shows an absorption peak typical of a mixture of LH2 and CC. The absorption spectra of the first peak, when analyzed using the extinction coefficients determined for the individual pigment protein complexes gives a CC:LH2 ratio of 1.5, this would suggest either some contamination due to poor separation from CC and/or some higher order oligomers containing more than one CC attached to an LH2.\n\nThe presence of relatively large amounts of monomeric proteins and several peaks of low molecular components with UV absorption was neither expected nor desired. The most likely explanation of this is poor separation of labeled protein from unreacted or click chemistry reagents coupled with poor yield in the reaction between the maleimides and the thiol-containing protein. Unfortunately we have not as yet been successful in addressing this yield issue. Nevertheless, the protocol we have developed allows to form and separate heterodimers of several different proteins in a controlled manner.\n\nWhile the polypeptides of certain LH2 complexes, for example those of Rb. sphaeroides or Rsb. denitrificans only contain lysine residues in the N-terminal, cytoplasmic, part of the sequence others such as those from Rps. acidophila or Phsp. molischianum have lysines in both the N and C terminal portions. Equally in core complexes there are lysines in the N terminal regions of the core antennae but also, depending on the species, various other potentially reactive lysines in the reaction center subunits and occasionally in the C-terminal region. To better understand the structure of the dimers formed, and assess their biological relevance, it would be useful to determine which lysines are linked together by cross-linking. Unfortunately our attempts to locate the labeling positions the labeling positions in the LH2 and core complexes from Phsp. molischianum, by MALDI-MS following digestion with various proteases (trypsin, chymotrypsin of V8 protease) were unsuccessful. Mass spectometry measurements were made on a Bruker Microflex II in positive reflectron mode with automatic sampling and peak identification followed by manual verification. The inability to determine the site of labeling in core complexes at low degrees of labeling, suggests perhaps that labeling is on the more numerous light harvesting polypeptides, since no difference was observed in the reaction center polypeptides detected before and after reaction even with degrees of labeling above 1. This however remains circumstantial and highly tentative.\n\n\nDiscussion\n\nThe general protocol we propose is able to produce purified dimeric, and possibly trimeric, complexes from the proteins of the purple bacterial photosynthetic apparatus. The approach based on reaction at a very low degree of labeling to ensure essentially mono-derivatized oligomers, despite the large number of equivalent reactive groups, followed by high selectivity and specificity purification of the activated proteins and copper-free click chemistry is able to overcome several of the problems we have previously encountered in trying to obtain such complexes. Notably the formation of highly cross-linked products is not observed, such multimeric products would be expected to elute earlier from the gel filtration column (4 min). The destruction of pigments inherent to copper (I) based cyclo-addition was also avoided.\n\nNevertheless the approach is not without problems and two are particularly flagrant. First the yield in the final step is much less than expected, and appears to be rather variable. Second we have also been unsuccessful in determining the site of labeling.\n\nThe low yield in the final step could derive from several different causes. First, the presence of contaminant DBCO and Azide after purification of the labeled protein, could lead to side reactions inactivating the labeled proteins. Second, the reactivity of the Cu free reagents could lead to deactivation/reaction before the mixing of the two proteins. Third, the maleimide labeling could be less efficient than expected. We suspect at least the first two possibilities are partly responsible, but have been unable to resolve the issue.\n\nThe difficulty in determining the site of labeling is the consequence of 2 different factors. On the one hand membrane proteins are often rather hard to study by mass spectrometry, and this results in low yields and coverage with proteomic approaches and difficulties in obtaining total mass due to the presence of detergents.\n\nThe site of labeling of the different proteins does however need to be confirmed to build models of such complexes and understand their behavior. This absence of knowledge is less important for certain complexes, for example as mentioned above the LH2 of Rsb. denitrificans has only two lysines in the N-terminal portion of the protein, α5 and β720, thus we can be fairly certain labeling is close to the cytoplasmic side of the protein. Unfortunately for the majority of complexes such simple deduction is not possible. However for the core complex we have some circumstantial indications that labeling is of the more abundant light harvesting polypeptides as we do not observe changes in the mass spectrum of the reaction center components at a degree of labeling of 1.0.\n\n\nConclusions\n\nWe have developed a novel protocol based on copper-free click chemistry that allows the formation and purification of specific dimers between highly oligomeric proteins. This protocol can be used to prepare biologically relevant dimers of certain LH2s with very asymmetrical lysine distribution, for example the LH2 of Roseobacter denitrificans or Rhodobacter sphaeroides. Such dimers will be of considerable interest for studying energy migration in light-harvesting arrays.\n\n\nData availability\n\nF1000Research: Dataset 1. Extinction coefficients for purified complexes, 10.5256/f1000research.8676.d12134921\n\nF1000Research: Dataset 2. Degree of labeling for different complexes, 10.5256/f1000research.8676.d12135022\n\nF1000Research: Dataset 3. HPLC trace after cross-linking, 10.5256/f1000research.8676.d12135123\n\nF1000Research: Dataset 4. Spectra of peaks in HPLC profile, 10.5256/f1000research.8676.d12135224",
"appendix": "Author contributions\n\n\n\nJS Designed the research, analyzed the data, prepared figures and wrote the article. WD Performed the experiments, analyzed the data and prepared figures.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing influences.\n\n\nGrant information\n\nThis work was funded by the China Scholarship Council (WD), the CNRS (JS) and Aix-Marseille University (JS).\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\nMass spectrometry performed in connection with this article was carried out in the facility of the Institute de Microbiology de la Mediterranée (IMM) by Pascal Manchot and Regine Lebrun.\n\n\nReferences\n\nLaw CJ, Roszak AW, Southall J, et al.: The structure and function of bacterial light-harvesting complexes. Mol Membr Biol. 2004; 21(3): 183–191. PubMed Abstract | Publisher Full Text\n\nScheuring S, Sturgis JN: Chromatic adaptation of photosynthetic membranes. Science. 2005; 309(5733): 484–7. PubMed Abstract | Publisher Full Text\n\nMcDermott G, Prince SM, Freer AA, et al.: Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature. 1995; 374(6522): 517–521. Publisher Full Text\n\nCogdell RJ, Gall A, Köhler J: The architecture and function of the light-harvesting apparatus of purple bacteria: from single molecules to in vivo membranes. Q Rev Biophys. 2006; 39(3): 227–324. PubMed Abstract | Publisher Full Text\n\nKoepke J, Hu X, Muenke C, et al.: The crystal structure of the light-harvesting complex II (B800–850) from Rhodospirillum molischianum. Structure. 1996; 4(5): 581–597. PubMed Abstract | Publisher Full Text\n\nSuzuki H, Hirano Y, Kimura Y, et al.: Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum. Biochim Biophys Acta. 2007; 1767(8): 1057–1063. PubMed Abstract | Publisher Full Text\n\nSturgis JN, Niederman RA: Atomic force microscopy reveals multiple patterns of antenna organization in purple bacteria: Implications for energy transduction mechanisms and membrane modeling. Photosynth Res. 2008; 95(2–3): 269–278. PubMed Abstract | Publisher Full Text\n\nSundstrom V, Pullerits T, van Grondelle R: Photosynthetic light-harvesting: Reconciling dynamics and structure of purple bacterial LH2 reveals function of photosynthetic unit. J Phys Chem B. 1999; 103(13): 2327–2346. Publisher Full Text\n\nMascle-Allemand C, Duquesne K, Lebrun R, et al.: Antenna mixing in photosynthetic membranes from Phaeospirillum molischianum. Proc Natl Acad Sci U S A. 2010; 107(12): 5357–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMascle-Allemand C, Duquesne K, Sturgis JN: Unpublished observations.\n\nDuquesne K, Blanchard C, Sturgis JN: Molecular origins and consequences of high-800 LH2 in Roseobacter denitrificans. Biochemistry. 2011; 50(31): 6723–6729. PubMed Abstract | Publisher Full Text\n\nSturgis JN, Hunter NC, Niederman RA: Spectra and extinction coefficients of near-infrared absorption bands in membranes of rhodobacter sphaeroides mutants lacking light-harvesting and reaction center complexes. Photochem Photobiol. 1988; 48(2): 243–247. Publisher Full Text\n\nClayton RK: Spectroscopic analysis of bacteriochlorophylls in vitro and in vivo. Photochem Photobiol. 1966; 5(8): 669–677. Publisher Full Text\n\nPapiz MZ, Prince SM, Howard T, et al.: The structure and thermal motion of the B800–850 LH2 complex from Rps.acidophila at 2.0A resolution and 100K: new structural features and functionally relevant motions. J Mol Biol. 2003; 326(5): 1523–38. PubMed Abstract | Publisher Full Text\n\nNiwa S, Yu LJ, Takeda K, et al.: Structure of the LH1-RC complex from Thermochromatium tepidum at 3.0 Å. Nature. 2014; 508(7495): 228–32. PubMed Abstract | Publisher Full Text\n\nSchrödinger LLC: The PyMOL molecular graphics system. Version 1.8. 2015. Reference Source\n\nvon Heijne G: Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol. 1992; 225(2): 487–494. PubMed Abstract | Publisher Full Text\n\nKhanna PL, Ullman EF: 4',5'-Dimethoxy-6-carboxyfluorescein: a novel dipole-dipole coupled fluorescence energy transfer acceptor useful for fluorescence immunoassays. Anal Biochem. 1980; 108(1): 156–161. PubMed Abstract | Publisher Full Text\n\nBaskin JM, Prescher JA, Laughlin ST, et al.: Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci U S A. 2007; 104(43): 16793–16797. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSwingley WD, Sadekar S, Mastrian SD, et al.: The complete genome sequence of Roseobacter denitrificans reveals a mixotrophic rather than photosynthetic metabolism. J Bacteriol. 2007; 189(3): 683–690. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSturgis J, Dong W: Dataset 1 in: Making dimers of oligomeric membrane proteins using copper free click-chemistry. F1000Research. 2016. Data Source\n\nSturgis J, Dong W: Dataset 2 in: Making dimers of oligomeric membrane proteins using copper free click-chemistry. F1000Research. 2016. Data Source\n\nSturgis J, Dong W: Dataset 3 in: Making dimers of oligomeric membrane proteins using copper free click-chemistry. F1000Research. 2016. Data Source\n\nSturgis J, Dong W: Dataset 4 in: Making dimers of oligomeric membrane proteins using copper free click-chemistry. F1000Research. 2016. Data Source"
}
|
[
{
"id": "14129",
"date": "06 Jun 2016",
"name": "Tatas Brotosudarmo",
"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 report of using click chemistry to label the protein of LH2 and core complex is interesting, however I have not been convinced by the data from spectroscopy nor biochemistry that can show the existence or the formation of a dimeric form of the LH2 or core complex polypeptides. The absorption spectra in Figure 4 resemble very much with the spectra of the native LH2 or core complex polypeptides. The B820 dimeric form was not there. CD spectrum might be a useful method to know.",
"responses": []
},
{
"id": "14131",
"date": "06 Jun 2016",
"name": "Kamil Woronowicz",
"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 lays a very important ground work and proof of principle for crosslinking photosynthetic complexes using click chemistry. It has already solved several major hurdles such as Cu-related loss of pigment color and similar effects due to reducing agent. It is using a very clever approach for low level modification and purification of these modified complexes using streptavidin beads, yet choosing to elute using DTT. It is somewhat surprising that TCEP, being a milder reducing agent, appears to be harsher on these pigmented complexes than DTT. It would also be great to see a comparison to betamercapthoethanol (BME), another well-known reducing agent, as well as an array of less known alternatives.\nNevertheless, the discussion identifies weaknesses of current work appropriately. These weaknesses could be addressed in a follow up work either by the Sturgis lab or anyone else following the precedent set by this work. Some additional comments might include poor resolution of size exclusion HPLC (Fig. 4A), though absorption spectra (Fig. 4B) show acceptable spectral purity, especially in context of the discussion presented by the author, exhibiting keen understanding of the nature of some of the impurities.\nIn order to address my reservations, I would like to see the following:\nThe paper mentions low yield of the last step, but doesn't provide quantitative data. Judging by the HPLC chromatogram in Fig. 4B it could be as much as 33%, but most likely it is much lower. If possible, either a Clear Native Gel or ultracentrifugation (especially analytical ultracentrifugation) might show relative amounts of complexes. Protein assay or amino acid analysis might be helpful in this regard as well.\n\nShowing normalized spectra shown in Fig. 4B (perhaps based on a ~600nm peak or one of the carotenoid peaks) could show the spectral shift and incorporation of both CCs and LH2s in the click-chemistry linked complex.\n\nProviding insight into yield using Cu. Even though the pigment is lost, but is the final step yield different (especially if significantly higher) as judged by HPLC? This could provide evidence that Cu-free system is not as efficient and might need additional improvements.\n\nBuilding on my previous remark, how optimized are current conditions? Have they been selected after a panel of unsuccessful trials or these represent the first or second trial? Specifically, are the temperatures and times of incubation at each step optimal? Click chemistry is typically thought of as almost quantitative, but it this very complex system there are several aspects to be considered that may decrease the yield.\n\nI think it would be great to see the HPLC of these protein complexes before and after to show appearance of a new peak corresponding to the linked complexes. It is unlikely that LH2 absorption would be observed in first peak since it shows up last as the peak representing the smallest complex. Unless, of course, it is present in such oligomeric form even before the reaction.\n\nLastly, I would like to see several editing corrections to be made:\na) page 4, in the paragraph that starts with \"Singly labeled proteins...\" second and third sentences should read \" The resulting proteins contain a unique reactive thiol, SINCE THE NATIVE proteins used do not have exposed cysteins. In the next step...\" new suggestion is shown in all CAPS, but does not need to be capitalized in the final text. Also, the word \"So\" has been removed from the beginning of the third sentence in this paragraph.\nb) continuing a few lines, instead of “each other” perhaps “a complimentarily modified counterpart” might be more suitable.\nc) Controlling degree of labeling paragraph, remove \"this\" in the second sentence.\nd) Same paragraph as part b) refer to Figure 1 when referring to \"extinction coefficients calculated as described above, and then check spelling of fluorescein immediately following.\ne) same paragraph, when talking about threshold, could you mention how many lysines do you expect to have available? It could be at least a measure of expected threshold.\nf) The second and third sentences under “Purifying low yield labeled proteins” I would paraphrase into “We chose to use succinimidyl-2-(biotinamido)ethyl-1,3,dithiopropionate, allowing strong and specific binding of labeled protein to streptaidin beads. Pure protein was eluted either with biotin or by reducing the disulfide bond”\ng) Next sentence I would take out “as expected”, and add “non-specific binding” when referring to contamination of unlabeled protein without biotin.\nh) It would be great to have a mention of what the 5% impurities are in the biotin-containing proteins (or sizes, or at least how that was established)\ni) Next paragraph, I would like to add “since” after the comma in the first sentence. Also, could you provide % purity or yields in support of your decision to use reducing agent?\nj) Same paragraph (third under Purifying low yield labeled proteins, middle right hand side page 4 on PDF, roughly in the middle of the page) very end: How did this gel filtration step on a spin column affect the impurities (see h above)? Would another SEC be needed to show that?\nk) Next paragraph, second sentence refer to nanomeric protein COMPLEX.\nl) Same paragraph, impressive statistical analysis of the distribution of modifications. Could you provide that as a supplementary material or show it somewhere?\nm) Last paragraph before discussion, about halfway: “the labeling positions” seems to be typed twice in a row.",
"responses": []
},
{
"id": "14130",
"date": "15 Jun 2016",
"name": "Robert A Niederman",
"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\nThese authors have attempted to develop a method for transforming the highly oligomeric light harvesting 2 (LH2) and LH1-reaction center (RC) core complexes into dimers (and trimers) of the alpha/beta hetrodimer, as a means of obtaining an improved understanding of energy transfer within isolated portions of the full arrays. In ¶4 of the Introduction, the authors need to cite and elaborate on the work by Westerhuis et al. (2002), stating that a method was previously developed for the isolation of a series of light-harvesting 1 (LH1) oligomers from the fully arrayed LH1-RC core complex. In this procedure, a Rhodobacter sphaeroides mutant lacking LH2 was subjected to lithium dodecyl sulfate polyacrylamide gel electrophoresis, which gave rise to a ladder of LH1 bands representing a series oligomers of the alpha/beta heterodimeric unit, varying in size from (alpha/beta )2-3 to (alpha/beta)10-11. Moreover, these oligomers exhibited oligomeric-state dependent optical properties, characterized by red shifts in near-IR absorption and emission maxima of ~6 nm at 77 K, as the aggregate sizes increased from 3 to 7-8 alpha/beta-heterodimers, accompanied by shifts in highly polarized fluorescence from the blue to the red side of the absorption band. This has been explained by the oligomerization of heterodimers to form a curvilinear array of excitonically-coupled chromophores, with an anisotropic long-wavelength component corresponding to low energy excitonic transitions arising from interactions within inhomogeneous BChl clusters.\nIn light of these findings, the authors can further justify why their method has been developed for the isolation of dimers and trimers of defined architecture in a controlled manner. But will much useful information come out of dimers and trimers when the full LH1 red shift required going up to an octameric state? The type of proposed energy migration studies planned for the isolated small arrays should be mentioned?\nRegarding Fig. 4, I don’t believe the source of these complexes has been designated. Fig.4A, is a second purification done to further purify these peaks? Fig. 4B, all three spectra need to be normalized at 590 nm to better show the composition of the cross-linked entities. The complex ratios can then be more precisely determined.\n\nOther errors found in text: P. 2, cloumn1, ¶2, line 6: Phaeospirillum (Phs.) molischianum P. 3, column 1, ¶2, line 14: Phs. molischianum, Rhodobacter (Rba.) P. 3, column 1, ¶2, line 1: of the complexes P. 3, column 1, 6 lines up dibenzylcyclooctyne (DBCO) P. 3, column 2, ¶2, line 4: (300 mm x 4.60 mm) P. 3, column 2, ¶3. line 3: Rhodoblastus (Rbl.) acidophilus, Phs. molischianum—2 lines down: Allochromatium (Ach.) P. 4, column 2, ¶4, 3 lines from bottom: 50 mM P. 5, column1, ¶1, line 1: 50-fold P. 5, column1, ¶2: Phs. molischianum omit “Phaeospirillum” P. 5, column 2, ¶1, line 2: Rba. sphaeroides, 2 lines down: Rbl. acidophilus, Phs. molischianum (and 12 lines up)\nP. 6, column1, ¶4: This ¶ needs to be corrected (2 different factors??) line 4 after: with proteomic approaches, add: and on the other hand, difficulties occur in obtaining ………\nIt should be noted that difficulties, making integral membrane proteins hard to study by mass spectroscopy arise by virtue of their hydrophobicity leading to a bias toward soluble hydrophilic peptides. The latter are more easily recovered during sample processing and separation, and ionize and dissociate better during mass spectroscopy. Moreover, some membrane proteins are insoluble under enzyme digestion conditions and can also precipitate during the subsequent analysis steps.",
"responses": []
},
{
"id": "14481",
"date": "20 Jun 2016",
"name": "Takehisa Dewa",
"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 described an approach to hetero-coupling of bacterial photosynthetic apparatuses, LH2 and core complex. I think their approach using copper-free Huisgen cyclization is reasonable. Although reaction yields are unfortunately very low, careful and patient chemistry could overcome these problems that they described. I had tried similar hetero-coupling reactions but it could not work well. I appreciate their work because of the descriptions about some trials, e.g., using TCEP/DTT.\nI like to suggest some points, for which I guess the authors have done experiments described below.\nSDS-PAGEs may clearly indicate which polypeptides reacted with . Reacted polypeptides (not only hetero-coupling products, but also NHS-SS-biotin adducts, DBCO- and Azide-bearing ones) may provide better MS after RP-HPLC purification.\n\nTo make sure the attachment and reactivity of DBCO and azide moieties, small molecules bearing azide and DBCO as their reaction counterparts should be useful. It may make clear whether DBCO and azide conjugates remain active.\n\nCharacterization using fluorescence spectroscopy is expected to see energy transfer from LH2 to cc.\n\nIt would be very helpful to understand the position of Lys if amino acid sequences of polypeptides of LH2 and cc used are listed.\nOther comments:\nIn Abstract: “56 individual polypeptides”\nWhere does this number come from?\nThe title is “making dimers”, but in Abstract description “making small oligomer, dimer and trimmers” is inconsistent. Major products seems dimer (heterodimer), so the description should be better to be just “dimer” to remove ambiguity.\n\npage 4, 5th paragraph (right column): Explanations for reaction levels are hard to understand. Descriptions “90.4% unlabeled protein” and “94.6% singly labeled protein” are inconsistent.\n\nFigure 4: This is one successful result showing the formation of LH2-cc hetero dimer. However, it is unclear what LH2 and cc were used. The authors used carious LH2 and cc from different photosynthetic bacteria. Origin of these complexes should be denoted.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1061
|
https://f1000research.com/articles/5-56/v1
|
12 Jan 16
|
{
"type": "Research Article",
"title": "Increasing the public health potential of basic research and the scientist satisfaction. An international survey of bioscientists",
"authors": [
"Giorgio Scita",
"Carmen Sorrentino",
"Andrea Boggio",
"David Hemenway",
"Andrea Ballabeni",
"Carmen Sorrentino",
"Andrea Boggio",
"David Hemenway"
],
"abstract": "Basic scientific research generates knowledge that has intrinsic value which is independent of future applications. Basic research may also lead to practical benefits, such as a new drug or diagnostic method. Building on our previous study of basic biomedical and biological researchers at Harvard, we present findings from a new survey of similar scientists from three countries. This survey asked about the scientists’ motivations, goals and perspectives along with their attitudes concerning policies designed to increase both the practical (i.e. public health) benefits of basic research as well as their own personal satisfaction. Close to 900 basic investigators responded to the survey; results corroborate the main findings from the previous survey of Harvard scientists. In addition, we find that most bioscientists disfavor present policies that require a discussion of the public health potential of their proposals in grants but generally favor softer policies aimed at increasing the quality of work and the potential practical benefits of basic research. In particular, bioscientists are generally supportive of those policies entailing the organization of more meetings between scientists and the general public, the organization of more academic discussion about the role of scientists in the society, and the implementation of a “basic bibliography” for each new approved drug.",
"keywords": [
"Basic research",
"Public health",
"Scientist satisfaction",
"Policy",
"Nudges",
"Grant",
"Basic bibliography"
],
"content": "Introduction\n\nBasic research has been crucial for the improvement of the human condition, including both research inspired solely by scientific curiosity and research driven by a vision of future applications. While basic knowledge is inherently valuable, all basic knowledge does not have the same potential for practical benefits. Of course, it is often difficult a priori as well as a posteriori to determine which knowledge will have, or has had, a greater impact on society, and some knowledge may never have any utility, neither direct nor indirect, in producing any practical outcome1–17. However, although we cannot know the future practical benefits of basic research, it is generally possible to make rough estimates of the potential.\n\nWe created a survey to assess the attitudes and beliefs of basic scientific researchers concerning policies that might incentivize bioscientists to engage in basic research with a higher likelihood of creating public health benefits, without compromising the “basic” nature of their research. We focused on policies based on soft incentives (what behavioral economists call “nudges”)18 because we believed that, if properly tailored to basic scientists motivations and goals19, soft policies could effectively stir (some) basic scientists towards research with a greater potential of creating larger public health benefits without overly constraining their research or decreasing their work satisfaction.\n\nTo determine which “nudges” might be effective, it is key to have a good grasp of what motivates basic scientists and of the intellectual framework in which they operate. To explore these questions, we previously conducted a study (Study 1) at a single institution (Harvard University and affiliated institutions in the Boston (MA-USA) area) to collect preliminary data and refine our hypotheses20. We found that the vast majority of the biological/biomedical scientists at Harvard University believe that, although is it often difficult to assess the potential future health benefits to society from basic research proposals, or actual research findings, some degree of estimation is possible. These bioscientists also supported the idea that softer policies are preferable to stricter ones for increasing the societal benefits of research.\n\nBased on the findings of Study 1, we designed this second study (Study 2) to obtain a larger sample of basic bioscientists, from multiple institutions and different countries. We used a modified version of the Study 1 questionnaire and added questions asking respondents to provide feedback concerning current policies and six new soft policies that we developed after analyzing the results of Study 1. In the current paper, we present the results of Study 2.\n\nJust under 900 basic bioscientists responded to the survey, completing an online questionnaire (see Methods section). Study 2 confirms the main findings of Study 1 with regard to motivations of basic scientists and how they conceptualize basic research. In particular, the vast majority of respondents reported being driven not only by curiosity or the desire of knowledge advancement but also by the aspiration of having an impact on people’s health. Respondents also think that basic scientists can ponder future practical benefits of their research without losing their “basic status” and that it is possible to roughly estimate the practical potential of basic research proposals. Finally, participants, especially principal investigators (PIs), disfavor current policies requiring the discussion of the potential societal impact in research proposals but favor the new policies we propose.\n\n\nMethods\n\nThe survey was an anonymous online questionnaire (see Questionnaire in the Data availability section) that was sent by email to scientists working at institutions where basic research in the biological/biomedical area is routinely conducted. The research instrument for Study 2 was a modified and expanded version of the questionnaire used in Study 1. In particular, we used a subset of the questions used in Study 1 and added questions on some current policies and on six policies that we propose.\n\nOver seven thousand (7,786) scientists were contacted from over thirty institutions in four different geographical locations [Los Angeles-San Diego (CA-USA), London-Cambridge (UK), Milan (Italy), and New York City (NY-USA)]. Invited scientists from the Los Angeles-San Diego area were affiliated with Calibr (California Institute for Biomedical Research), Caltech (California Institute of Technology), Cedars-Sinai, Salk Institute, Sanford-Burnham Medical Research Institute, Scripps Research Institute, UCI (University of California Irvine), UCLA (University of California Los Angeles), UCR (University of California Riverside), UCSD (University of California San Diego) or USC (University of Southern California). Invited scientists from the London-Cambridge area were affiliated with Francis Crick Institute, ICL (Imperial College London), ICR (Institute of Cancer Research), King’s College, UCL (University College London), University of London Birkbeck, University of London Queen Mary, University of London St George’s or the University of Cambridge. Invited scientists from the Milan area were affiliated with Humanitas Research Hospital, IEO (European Institute of Oncology), IFOM (FIRC Institute of Molecular Oncology), INGM (National Institute of Molecular Genetics), Istituto Nazionale dei Tumori (National Institute of Tumors), Mario Negri Institute, San Raffaele Hospital, University of Milan or University of Milan-Bicocca. Invited scientists from the New York City area were affiliated with Albert Einstein College of Medicine, Columbia University, CSHL (Cold Spring Harbor Laboratories), CUNY ASRC (City University of New York Advanced Science Research Center), CUNY Queens (City University of New York Queens college), Memorial Sloan Kettering Cancer Center, Mount Sinai Hospital, NYU (New York University) or Rockefeller University.\n\nEmail addresses were taken from the publicly accessible websites of the institutes. Invitations were sent to individual emails. The invitations contained a standard text of invitation and brief explanation of the study; emails of invitations differed one from another only with regard to the name of the invited scientist in the salutation (“Dear xxx”).\n\nParticipation in the study was voluntary and entailed answering an online survey powered by Qualtrics software (www.qualtrics.com) through the Harvard T.H. Chan School of Public Health. Respondents could skip any questions they wanted to. They were asked to confirm their status as basic researchers (see Results section). We invited all types of bioscientists, with regard to their position/role, except that we tried not to invite undergraduate students and non-PhD technicians.\n\nWe tried to keep the proportion of invitations sent to PIs at over 50%. We did not invite every single scientist at every institute but for each institute decided a priori the number of scientists and then used the alphabetical order. Respondents had the option of indicating their geographical location but not their institution. The differences in gender invitations were not deliberate but a reflection of the actual proportions of females and males. Graphs describing the statistics of the invitations are shown in Figures S1–S6.\n\nThe invitations were sent from August 24, 2015 to October 10, 2015. Force completion of the survey was set at 72 hours. The study was reviewed and approved by the Harvard T.H. Chan School of Public Health IRB (IRB15-2787) and by the FIRC Institute of Molecular Oncology Ethics Committee.\n\n\nResults\n\n\n\n\nOverview of the sample\n\nClose to 900 (885) scientists responded to the survey. The overall response rate was 11.4%. The response rates for females and males were 12.8% and 10.0%, respectively (the response rates for females were higher in all geographical locations). The response rate for PIs was 10.5% (12.4% for female PIs and 9.6% for male PIs). Detailed response rates are shown in Figures S7–S11. There were 464 respondents who reported PI status, 219 post-docs, 94 PhD students and 109 other/unspecified roles.\n\nMore males (500) than females (359) participated in the study. The average age was 43.4 years old. 202 worked in the Los Angeles-San Diego area (CA-USA), 180 in the London-Cambridge area (UK), 223 in the Milan area (Italy), and 238 in the New York City area (NY-USA). From the question “Approximately, what percentage of your research do you consider to be basic?” the average level of involvement in basic research was 78.0% (2.7% of the respondents skipped this specific question). Only 3 respondents (0.3%) declared they were not involved in basic research at all (i.e. 0% of basic research). Questions were skipped in the range of 2.3% (question with the lowest skipping rate) to 13.7% (question with the highest skipping rate). The number of responses according to role, gender, geographical location and level of involvement in basic research are shown in Figures S12–S18. All responses are presented in the Final report in the Data availability section.\n\n\nThe motivations of the basic scientists\n\nWe asked participants to rank their motivations for research by level of importance. “Health benefit to society”, “satisfaction of curiosity”, and “satisfaction from solving puzzling problems” were the most important motivations while “gain of prestige” and “gain of money” were less important motivations. “Pure advancement of knowledge” was a strong motivator, especially among principal investigators. These results confirmed the findings of Study 1 (Figures 1a–f) (detailed data for all figures in this paper are shown in the associated tables in Supplementary material).\n\n(a) Pure advancement of knowledge, regardless of future applicability, (b) Health benefit to society (not necessarily in the near future), (c) Gain of prestige, (d) Gain of money (for personal purposes), (e) Satisfaction of your curiosity, (f) Satisfaction from solving puzzling problems.\n\nFor PIs, their level of involvement in basic research was positively correlated with the motivations “pure advancement of knowledge,” “satisfaction of curiosity,” and “satisfaction from solving puzzling problems” and negatively correlated with the motivations “health benefit to society,” “gain of prestige,” and “gain of money” (Figures 2a–f).\n\n(a) Pure advancement of knowledge, regardless of future applicability, (b) Health benefit to society (not necessarily in the near future), (c) Gain of prestige, (d) Gain of money (for personal purposes), (e) Satisfaction of your curiosity, (f) Satisfaction from solving puzzling problems. (Principal Investigators ordered by percentage of basic research).\n\n\nThe concept of basic research and its practical benefit potential\n\nThe vast majority of the surveyed scientists were in some or complete agreement with the statement: “basic scientists can ponder about the future indirect practical benefits of their research without losing their ‘basic status’” (Figure 3). The majority indicated that the most important goal of publicly funded basic biological research should be “pure advancement of knowledge, regardless of future applicability” (Figure 4a) and of funded basic biomedical research should be the “health benefit to society (not necessarily in the near future)” (Figure 4b). PIs with more involvement in basic research were more likely to agree with the statement “basic scientists can ponder about the future indirect practical benefits of their research without losing their ‘basic status’” (Figure 5) and that “pure knowledge advancement” is the main goal of basic research (Figures 6a–b).\n\nPrincipal investigators ordered by percentage of basic research.\n\n(Principal investigators ordered by percentage of basic research).\n\n\nThe policy of discussing health benefits in research proposals\n\nOver 70% of respondents expressed at least some agreement with the statement: “although it is difficult to assess the potential future health benefits to society from basic biological/biomedical research as described in written proposals, some degree of estimation is always possible” (Figure 7). However, the level of agreement was significantly lower with the statement: “written proposals about basic biological/biomedical research generally contain a section discussing potential future health benefits. These sections increase the likelihood that a project benefits future public health.” The difference was especially important for PIs, who expressed the highest degree of disagreement with the second statement (Figure 8).\n\nAlmost half of respondents agreed with the statement that “writing the sections discussing potential future health benefits takes too much time” (Figure 9a). For the statement “the sections discussing potential future health benefits should be eliminated for [no/a few/most/all] grants”, over 70% of respondents declared that these sections should be eliminated at least for some grants, including a significant portion (especially of PIs) that indicated that these sections should be eliminated for “most” or “all” grants (Figure 9b).\n\nFor PIs, there was a negative correlation between their level of involvement in basic research and their level of agreement with the statement “although it is difficult to assess the potential future health benefits to society from basic biological/biomedical research as described in written proposals, some degree of estimation is always possible” although most PIs, even those with the highest involvement in basic research, were in agreement (Figure 10). There was also a negative correlation between the degree of involvement in basic research and the statement “written proposals about basic biological/biomedical research generally contain a section discussing potential future health benefits. These sections increase the likelihood that a project benefits future public health.” Here, over two thirds of the PIs with the highest involvement in basic research were in disagreement (Figure 11).\n\nPrincipal Investigators ordered by percentage of basic research.\n\nPrincipal investigators ordered by percentage of basic research.\n\nAmong PIs, more involvement in basic research led to more support for the statement “writing the sections discussing potential future health benefits takes too much time” (Figure 12a) and more support for the idea that “the sections discussing potential future health benefits” should be eliminated for at least a subset of grants. This latter opinion was expressed by fewer than 50% of PIs with the least involvement in basic research but by almost 80% of PIs with the highest involvement in basic research (Figure 12b).\n\nPrincipal investigators ordered by percentage of basic research.\n\nOverall, the results show that most of basic scientists believe that some degree of assessment of the health benefit potential of basic biological or biomedical research is possible but that the current policy requiring the discussion of this potential in written research proposals is not very effective and should be eliminated for at least a portion of the grants, if not most or all of them.\n\n\nSoft policies to increase the public health potential of basic research and the satisfaction of scientists\n\nWe tested scientists’ opinions concerning six soft policies that we designed based on the results of Study 1. These policies are intended to increase the public health potential of basic investigations and improve the work satisfaction of the basic scientists.\n\nThese are the policies:\n\nA. “Locate more basic research laboratories inside or in close proximity of hospitals.”\n\nB. “Organize more educational and discussion meetings between scientists and the general public or patient associations. Acknowledge participating scientists during grant assignments, promotion, hiring etc.”\n\nC. “Promote more seminars and academic discussion concerning the purpose of scientific research and the role of scientists in the society. Acknowledge participating scientists during grant assignments, promotion, hiring etc.”\n\nD. “Promote more seminars and academic discussion about the concept and definition of basic research. Acknowledge participating scientists during grant assignments, promotion, hiring etc.”\n\nE. “Have ethics consultation services for scientists inside research institutes, with easily accessible information about these services.”\n\nF. “Provide recognition to basic scientists who have contributed to acquiring key knowledge that leads to tangible health benefits by requiring a \"basic bibliography\" of seminal basic research articles for each new drug or other biological application.”\n\nRespondents were asked to evaluate these policies on four criteria: the policy’s effectiveness in generating (a) societal benefit, (b) scientists’ work satisfaction, along with the policy’s (c) feasibility and (d) overall favorability. Respondents were asked to evaluate these policies using four scores: “none” (score 1), “low” (score 2), “medium” (score 3), “high” (score 4). The vast majority of scientists judged all six policies to have at least some degree of effectiveness, feasibility and favorability, with a substantial proportion of scientists giving scores of 3 or 4. The policies that scored highest with regard to the societal benefit were B (score 3.3), C (score 3.2) and F (score 3.2). The policy that scored highest with regard to the scientist satisfaction was F (score 3.4). The policies that scored highest with regard to feasibility were F (score 3.2), C (score 3.1) and E (score 3.1). The policies that scored highest with regard to overall favorability were F (score 3.3), B (score 3.2) and C (score 3.2). Policy F had the highest percentage of “high” responses (scores 4) (46.5%) and the highest percentage of “high” responses in terms of overall favorability (49.9%) (Figures 13a–f). Role of the respondent (i.e., PI, post-doc) did not substantially affect the favorability of these options (Figures 14a–f).\n\n(a) “Locate more basic research laboratories inside or in close proximity of hospitals”. (b) “Organize more educational and discussion meetings between scientists and the general public or patient associations. Acknowledge participating scientists during grant assignments, promotion, hiring etc.” (c) “Promote more seminars and academic discussion concerning the purpose of scientific research and the role of scientists in the society. Acknowledge participating scientists during grant assignments, promotion, hiring etc.” (d) “Promote more seminars and academic discussion about the concept and definition of basic research. Acknowledge participating scientists during grant assignments, promotion, hiring etc.” (For example, should basic research be conceptualized as purely curiosity-driven, or could basic scientists also consider future indirect practical benefits of their research?). (e) “Have ethics consultation services for scientists inside research institutes, with easily accessible information about these services”. (f). “Provide recognition to basic scientists who have contributed to acquiring key knowledge that leads to tangible health benefits by requiring a “basic bibliography” of seminal basic research articles for each new drug or other biological application”.\n\n(a) Favorability for “Locate more basic research laboratories inside or in close proximity of hospitals”. (b) Favorability for “Organize more educational and discussion meetings between scientists and the general public or patient associations. Acknowledge participating scientists during grant assignments, promotion, hiring etc.” (c) Favorability for “Promote more seminars and academic discussion concerning the purpose of scientific research and the role of scientists in the society. Acknowledge participating scientists during grant assignments, promotion, hiring etc.” (d) Favorability for “Promote more seminars and academic discussion about the concept and definition of basic research. Acknowledge participating scientists during grant assignments, promotion, hiring etc.” (For example, should basic research be conceptualized as purely curiosity driven, or could basic scientists also consider future indirect practical benefits of their research?). (e) Favorability for “Have ethics consultation services for scientists inside research institutes, with easily accessible information about these services”. (f) Favorability for “Provide recognition to basic scientists who have contributed to acquiring key knowledge that leads to tangible health benefits by requiring a “basic bibliography” of seminal basic research articles for each new drug or other biological application”.\n\nFor PIs, in terms of degree of involvement in basic research, there were substantial differences in favorability only in relation to policy A, for which there was a small negative correlation (Figures 15a–f). With regard to the influence of the gender of the PIs, there was a slight increase in favorability of female PIs for policy B–F and in favorability of male PIs for policy A (Figures 16a–f).\n\n(a) Favorability for “Locate more basic research laboratories inside or in close proximity of hospitals”. (b) Favorability for “Organize more educational and discussion meetings between scientists and the general public or patient associations. Acknowledge participating scientists during grant assignments, promotion, hiring etc.” (c) Favorability for “Promote more seminars and academic discussion concerning the purpose of scientific research and the role of scientists in the society. Acknowledge participating scientists during grant assignments, promotion, hiring etc.” (d) Favorability for “Promote more seminars and academic discussion about the concept and definition of basic research. Acknowledge participating scientists during grant assignments, promotion, hiring etc.” (For example, should basic research be conceptualized as purely curiosity driven, or could basic scientists also consider future indirect practical benefits of their research?). (e) Favorability for “Have ethics consultation services for scientists inside research institutes, with easily accessible information about these services”. (f) Favorability for “Provide recognition to basic scientists who have contributed to acquiring key knowledge that leads to tangible health benefits by requiring a “basic bibliography” of seminal basic research articles for each new drug or other biological application”. (Principal Investigators ordered by percentage of basic research).\n\n(a) Favorability for “Locate more basic research laboratories inside or in close proximity of hospitals”. (b) Favorability for “Organize more educational and discussion meetings between scientists and the general public or patient associations. Acknowledge participating scientists during grant assignments, promotion, hiring etc.” (c) Favorability for “Promote more seminars and academic discussion concerning the purpose of scientific research and the role of scientists in the society. Acknowledge participating scientists during grant assignments, promotion, hiring etc.” (d) Favorability for “Promote more seminars and academic discussion about the concept and definition of basic research. Acknowledge participating scientists during grant assignments, promotion, hiring etc.” (For example, should basic research be conceptualized as purely curiosity driven, or could basic scientists also consider future indirect practical benefits of their research?). (e) Favorability for “Have ethics consultation services for scientists inside research institutes, with easily accessible information about these services”. (f) Favorability for “Provide recognition to basic scientists who have contributed to acquiring key knowledge that leads to tangible health benefits by requiring a “basic bibliography” of seminal basic research articles for each new drug or other biological application”. (Principal Investigators and Gender).\n\n\nConclusions and discussion\n\nTen main results deserve some emphasis:\n\n• Basic scientists are strongly motivated not only by “satisfaction of curiosity” and “from solving puzzling problems” but also by the possible future practical benefits to society.\n\n• There is a positive correlation between involvement in basic research and the importance of motivation from “pure advancement of knowledge,” “satisfaction of curiosity,” and “satisfaction from solving puzzling problems.”\n\n• There is a negative correlation between involvement in basic research and motivation for the “health benefit to society.”\n\n• PIs are more motivated than other types of investigators from “pure advancement of knowledge,” “satisfaction of curiosity,” and “satisfaction from solving puzzling problems.”\n\n• While money is not a powerful motivation, prestige is an important to moderately important motivation for nearly half of PIs.\n\n• Almost all scientists think that it is possible to ponder possible future applications of basic investigations without compromising the basic nature of the research.\n\n• On the other hand, most basic scientists disfavor policies mandating the discussion of these practical applications in research proposals.\n\n• There is a positive correlation between PIs’ involvement in basic research and their rejection of the requirement of discussing the health benefit potential in research proposals.\n\n• There is a large consensus on the effectiveness, feasibility and favorability for all six soft policies designed to increase the public health potential of basic research and the scientist satisfaction.\n\n• Among these six policies, those entailing the organization of more meetings between scientists and the general public, the organization of more academic discussion about the role of scientists in the society, and the implementation of a “basic bibliography” for each new approved drug received the highest approval rates.\n\nThis study is a follow-up to our previous study of a smaller sample of basic scientists working at a single institution (Harvard University) in a single geographical area (the Boston area in the United States)20. Based on the results of that survey, we expanded the study to include questions on six specific soft policies and a larger sample of scientists working at different institutions in four geographical areas (see Figures S19–S26 for differences between locations) in three countries.\n\nThe current study confirmed much of what we had discovered in Study 1 with regard to basic scientists’ motivations and conceptualization of basic research. The current study also confirmed that, while some estimate of the public health potential of basic investigations is always possible, basic scientists believe that the requirement of discussing this potential in research proposals is not effective and should be eliminated for at least a portion of the grants (see also Figures S27–S30). The six proposed “nudge”-based policies were judged positively in terms of their future public health impact, scientist work satisfaction, and feasibility, with policies B, C and F receiving the highest approval ratings.\n\nThis study has several strengths. First, we gathered data from a large and diverse population of basic scientists. Second, we analyzed responses not only with respect to the role of the scientist (e.g., PI, post-doc) but also with the self-reported (from 0 to 100%) level of involvement with basic research. As far as we could determine, this has not been done before. Third, the survey has a depth and level of detail rarely seen in surveys of the motivations and perspectives of biological and biomedical scientists. Finally, and most important, this study provides information on specific policies, some of these new policies (B, C, D, F), and the findings can be used by policymakers to improve the governance of basic research.\n\nThe study has also weaknesses. First the survey results may not be fully representative of the views of all basic biomedical and biological scientists. Although based on a large and multinational sample, it still only presents the views of scientists in three countries. Moreover, since we focused our study mainly on PIs and post-docs, this study is less representative of the opinions of students or other types of scientists such as staff scientists or research technicians. In addition, although we had a large sample, only 11% of possible respondents answered the survey. Finally, although most of the surveyed scientists were positive about the effectiveness and feasibility of the proposed policies, that alone does not ensure these policies will in fact be effective and/or feasible. Similarly, it does not imply that other parts of the society (e.g. the general public and the policymakers) have the same views.\n\nWe hope the information and discussion provided in this paper will be useful to scholars, policymakers and advocates. We encourage them to foster the discussion and work for the implementation of policies that can benefit both society and science. The results provided in this paper suggest that the proposed policies are well grounded in the motivations and perspectives of the basic bioscientists and have their approval. We believe this is an important asset with respect to what would be the actual effectiveness of these policies and the potential for implementation.\n\n\nData availability\n\nF1000Research: Dataset 1. Questions and responses of the survey, 10.5256/f1000research.7683.d11088821",
"appendix": "Author contributions\n\n\n\nStudy design: Andrea Ballabeni, David Hemenway, Giorgio Scita. Data collection: Andrea Ballabeni, Carmen Sorrentino. Data analysis: Andrea Ballabeni, Andrea Boggio, David Hemenway, Carmen Sorrentino, Giorgio Scita. Manuscript writing: Andrea Ballabeni, Andrea Boggio, David Hemenway, Giorgio Scita.\n\n\nCompeting interests\n\n\n\nThe authors declare no competing interests.\n\n\nGrant information\n\nAndrea Ballabeni and Carmen Sorrentino were funded by Cariplo Foundation (grant #2015.0081).\n\nWe confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Celeste Ungaro (IFOM) for her administrative and organizational support, Assunta Croce (IFOM) for her feedback during the preparation of the study, Ivan Lago (IFOM) and Manuelo Malizia (IFOM) for their IT support. We thank Cariplo foundation (http://www.fondazionecariplo.it/en/index.html) for funding the study and Silvia Pigozzi (Cariplo foundation) for her assistance on behalf of the funding organization.\n\n\nSupplementary material\n\nFigures S1–S6. Data on the invitations to the questionnaire.\n\nFigures S7–S11. Data on the response rates of the questionnaire.\n\nFigures S12–S18. Data describing the sample of scientists participating to the questionnaire.\n\nFigures S19–S26. Data describing the differences in the responses based on geographical location.\n\nFigures S27–S30. Additional data on scientists views on criteria of evaluation and funding of basic research.\n\nTables showing the detailed percentages of responses for all the figures of the paper (Figures 1–16).\n\n\nReferences\n\nBallabeni A, Boggio A, Hemenway D: Recognizing Basic Science Contributions. The Scientist. 2014; 28: 26–27. Reference Source\n\nBeckwith J, Huang F: Should we make a fuss? A case for social responsibility in science. Nat Biotechnol. 2005; 23(12): 1479–1480. PubMed Abstract | Publisher Full Text\n\nBornmann L: Measuring the societal impact of research: research is less and less assessed on scientific impact alone--we should aim to quantify the increasingly important contributions of science to society. EMBO Rep. 2012; 13(8): 673–676. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBush V: Science the endless frontier. Trans Kans Acad Sci (1903-). Washington DC: US Government Printing Office. 1945; 48(3): 231–264. Publisher Full Text\n\nChalmers I, Bracken MB, Djulbegovic B, et al.: How to increase value and reduce waste when research priorities are set. Lancet. 2014; 383(9912): 156–165. PubMed Abstract | Publisher Full Text\n\nKuhn T: The Structure of Scientific Revolutions. 1962. Reference Source\n\nLadd JM, Lappé MD, McCormick JB, et al.: The \"how\" and \"whys\" of research: life scientists' views of accountability. J Med Ethics. 2009; 35(12): 762–767. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMerton R: The Sociology of Science: theoretical and empirical investigations. The University of Chicago Press. 1973. Reference Source\n\nSampat BN: Mission-oriented biomedical research at the NIH. Res Policy. 2012; 41(10): 1729–1741. Publisher Full Text\n\nStokes D: Pasteurs Quadrant: Basic Science and Technological Innovation. Washington DC: Brookings Institution Press. 1997. Reference Source\n\nWilsdon J, Wynne B, Stilgoe J: The public value of science: or how to ensure that science really matters. London: Demos, 2005. Reference Source\n\nPouliot C, Godbout J: Thinking outside the 'knowledge deficit' box. EMBO Rep. 2014; 15(8): 833–835. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRull V: The most important application of science: As scientists have to justify research funding with potential social benefits, they may well add education to the list. EMBO Rep. 2014; 15(9): 919–922. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcCormick JB, Boyce AM, Ladd JM, et al.: Barriers to Considering Ethical and Societal Implications of Research: Perceptions of Life Scientists. AJOB Prim Res. 2012; 3(3): 40–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBesley JC, Oh SH, Nisbet M: Predicting scientists' participation in public life. Public Underst Sci. 2013; 22(8): 971–987. PubMed Abstract | Publisher Full Text\n\nDubochet J: Teaching scientists to be citizens. It is hard to become a good scientist. It is even harder to become a good citizen. EMBO Rep. 2003; 4(4): 330–332. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaunders C, Girgis A, Butow P, et al.: Beyond scientific rigour: funding cancer research of public value. Health Policy. 2007; 84(2–3): 234–242. PubMed Abstract | Publisher Full Text\n\nThaler RH, Sunstein CR: Nudge: improving decisions about health, wealth, and happiness. New Haven: Yale University Press, 2008. Reference Source\n\nLam A: What motivates academic scientists to engage in research commercialization: ‘Gold’, ‘ribbon’ or ‘puzzle’? Res Policy. 2011; 40(10): 1354–1368. Publisher Full Text\n\nBallabeni A, Boggio A, Hemenway D: Policies to increase the social value of science and the scientist satisfaction. An exploratory survey among Harvard bioscientists [version 2; referees: 2 approved]. F1000Res. 2014; 3: 20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScita G, Sorrentino C, Boggio A, et al.: Dataset 1 in: Increasing the public health potential of basic research and the scientist satisfaction. An international survey of bioscientists. F1000Research. 2016. Data Source"
}
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[
{
"id": "13139",
"date": "01 Apr 2016",
"name": "Nathan L. Vanderford",
"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\nScita et al. present survey research findings on basic scientists’ perceptions of the public health relevance of their work as well as their attitudes toward their own personal satisfaction. This work is interesting especially as it relates to scientists’ perceptions of their work versus the public’s consumption of that work and the associated policies around that interaction.There are several issues with the current version of the article that lead me to approving it with reservations. The current title could be improved and clarified. The part on scientists’ satisfaction seems to be an afterthought as written. The specific research questions and/or hypotheses for the study should be clearly stated. Clarification is needed to allow readers to better understand the differences and similarities between “study 1” versus “study 2.” What is the overlap of the study design and findings? A better explanation as to how “study 1” informed “study 2” would be helpful. The data on the respondent demographics and response rates should be clarified (the current figures and text are not clear). Adding this information into the main text, in table format, may be useful. The clarity of the article would benefit from simplifying the presentation of the data: 16 figures in the main text is overwhelming. Can some of the data be summarized and collapsed into a couple of tables and/or can some of the data be included as supplemental material? The findings would be greatly enhanced by including some statistical testing. Related to that, what is the primary outcome variable(s) of the study? What are the independent and dependent variables? Testing for association of the outcome(s) with items such as career stage (eg, PI, postdoc, etc.), gender, age, and geographic location would be very interesting and informative. At the beginning of the soft policies section on page 12 of the PDF, the statement “[w]e tested scientists’ opinions…” should be demonstrated through statistical analysis. Without statistical analysis, the conclusions should not be written to suggest negative or positive correlations. The results of this study should be discussed in context of the related literature in the “conclusions and discussion” section. Perhaps it would be best to have a separate conclusion and discussion section. Why is it that the text in some of the figure legends is underlined and in other figure legends the text is not underlined?I look forward to reviewing a revised version of the article.",
"responses": [
{
"c_id": "1984",
"date": "01 Jun 2016",
"name": "Andrea Ballabeni",
"role": "Author Response",
"response": "We would like to thank Dr. Nathan Vanderford for reviewing our manuscript and for providing helpful suggestions and comments. We have revised the paper according to his feedback. We summarize here our responses, point by point We have changed the title to better emphasize that we aim at increasing both the public health potential and the work satisfaction. We have now inserted a new sentence in the abstract to clearly define the goals of the study. In the Introduction section we have edited the previous wording and this is the new wording: “We used a slightly modified version of the Study 1 questionnaire. In particular, we added a few questions asking respondents to evaluate current policies used to evaluate/increase the public health potential as well as six new soft policies that we developed after analyzing the results of Study 1.” In the Discussion section we have changed the text and this is the new wording: “The current study substantially confirmed what we had discovered in Study 1. In particular, the new study confirmed what we had previously observed with regard to basic scientists’ motivations and conceptualization of basic research. Moreover, it further reinforces the notion that, while some estimate of the public health potential of basic investigations is always possible, basic scientists believe that the requirement of discussing this potential in research proposals is not effective”. Moreover, in the Introduction section we have also added the following revised text: “Study 1 showed that increasing people’s health and personal prestige are some of the strongest motivations for basic scientists. Moreover, it showed that basic scientists strongly support the idea of non-mandatory policies based on soft incentives to increase public health potential and work satisfaction. Based on these and other findings of Study 1, we designed this second study (Study 2)…”. We would prefer to have the figures about the invitations (Figures S1-S6) and about the response rates (Figures S7-S11) in the “Supplementary material” section because not the main goals of our research. The manuscript appear already dense in information and we would like to include in the main text only the most relevant set of information related to the analysis of the responses. In addition, the “Overview of the sample” paragraph in the “Results” section already provides a summary of the sample and response rates. However, if the reviewer thinks that some additional information should necessarily be placed in the main text we would be happy to consider it. We agree with the reviewer that 16 figures are more than the conventional research articles contain and this is also the reason why we would avoid adding more figures or tables (e.g. for sample description and response rates) in the main text. The flexible format offered by F1000Research actually encourages the inclusion of figures and graphs. Based on these guidelines, we chose to show the data and convey the messages more through the figures than through the text. Indeed, even if the figures (which are not particularly dense in information and relatively easy-to-read bar/column charts) are more than in the standard average research article, the length of the text is probably shorter than in the average published manuscript. We have now performed statistical analysis on the association of the motivations (Figure 1) with the role of the scientists (PIs, post-docs, students). We have introduced the following new text: “For the motivations we also performed analyses based on the roles of the scientists. We observed that some differences were statistically significant. In particular, PIs are more motivated from “Pure advancement of knowledge” (p = 0.0042) and less motivated from “Gain of money” (p<0.0001) in comparison to post-docs. Moreover, PIs are more motivated from “Pure advancement of knowledge” (p<0.0001) and “Satisfaction of curiosity”(p = 0.0068) and less motivaed from “Gain of money” (p = 0.0142) in comparison to students.” We are not sure about the problem but, after careful consideration we think that the use of the term “tested” is appropriate in this context. There was indeed one point in which we suggested a negative correlation without providing statistical analysis. We have now performed the statistical analysis and amended the text as it follow: “For PIs, in terms of degree of involvement in basic research, there were substantial differences in favorability only in relation to policy A, for which there was a small negative correlation (R2 = 0.986) ( Figures 15a–f)” As suggested by the reviewer, we have separated the Conclusions from the Discussion section. Since the length of the manuscript and the emphasis on figures and results, we would prefer not add more text. However, we have added a note in the Discussion section to inform that an in-depth analysis of the relevant literature as well as a discussion on the conceptual framework are present inside the manuscript describing the results of the first study recently published (i.e. Study 1) (ref. 20). In some of the figure legends the text is underlined because we wanted to emphasize the specific policies. However, we agree that in this way the text is not easily readable. Therefore, we have accordingly modified the figure legends of Figures 13-16. We hope we have addressed all the reviewer’s comments and suggestions. We would like to thank Dr. Nathan Vanderford one more time for taking the time to review the manuscript and provide his useful feedback and we look forward to hearing from Dr. Vanderford."
}
]
},
{
"id": "13079",
"date": "05 Apr 2016",
"name": "Viviane Callier",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 research paper reports the results of a survey of biomedical and biological scientists about their motivations and attitudes with respect to basic research. They found that basic scientists are mainly driven by a goal to satisfy their curiosity and to advance fundamental knowledge. They also have an aspiration to impact human health, usually in an abstract or yet-undefined way. Prestige is a motivator for some.The thoroughly reported survey results showed that researchers feel ambivalent about the \"statement of public health relevance\" sections that accompany most grant proposals to biomedical funding agencies. Some favor getting rid of this section. This is important information for the scientific community and for funding agencies to consider.This research paper is timely because the importance of basic research has been on the mind of the leaders at NIH, the largest biomedical funding agency in the world, as shown in their recent letter to Science. In it, the NIH leadership says that the research community erroneously believes that the \"statement of public health relevance\" means that the agency does not value basic research, when in fact, the NIH leadership says it does. However, at several agencies including NINDS and NCI, the proportion of basic science proposals received is going down, while the proportion of \"hybrid\" or applied/clinical research proposals is going up. This means that many investigators are shifting their application strategy towards applied research because they think it is more likely to gain funding.Ultimately, it is the scientists on review panels, not just NIH leadership, who determine which research gets funded. So, if the agency and the research community values basic research, there needs to be a change in culture so that scientifically sound and meritorious proposals gain funding even if they do not have specific clinical applications. Reviewers in basic science study sections should be instructed to evaluate proposals on experimental design and novelty but not clinical relevance.Future research on how the tight funding climate has impacted attitudes towards basic research, and ideas on how to reverse the trend of decreasing numbers of basic research proposals, would be very interesting to funding agencies and policy makers.This is an interesting and well written paper. I have a few suggestions to improve the paper. There are many figures and the information contained in these figures could be consolidated into fewer figures to make it easier to interpret. Statistical analysis of the data would also make the conclusions stronger. All in all, the findings make intuitive sense and it is nice to have the data to confirm these intuitions.",
"responses": [
{
"c_id": "1985",
"date": "17 May 2016",
"name": "Andrea Ballabeni",
"role": "Author Response",
"response": "We would like to thank Dr. Viviane Callier for taking the time to read and review our manuscript. We have appreciated reading her thoughts on these topics."
}
]
}
] | 1
|
https://f1000research.com/articles/5-56
|
https://f1000research.com/articles/5-416/v1
|
30 Mar 16
|
{
"type": "Antibody Validation Article",
"title": "Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle",
"authors": [
"Christine Chaponnier",
"Giulio Gabbiani"
],
"abstract": "Higher vertebrates express six different highly conserved actin isoforms that can be classified in three subgroups: 1) sarcomeric actins, α-skeletal (α-SKA) and α-cardiac (α-CAA), 2) smooth muscle actins (SMAs), α-SMA and γ-SMA, and 3) cytoplasmic actins (CYAs), β-CYA and γ-CYA. The variations among isoactins, in each subgroup, are due to 3-4 amino acid differences located in their acetylated N-decapeptide sequence. The first monoclonal antibody (mAb) against an actin isoform (α-SMA) was produced and characterized in our laboratory in 1986 (Skalli et al., 1986). We have further obtained mAbs against the 5 other isoforms. In this report, we focus on the mAb anti-α-SKA and anti-α-CAA obtained after immunization of mice with the respective acetylated N-terminal decapeptides using the Repetitive Immunizations at Multiple Sites Strategy (RIMMS). In addition to the identification of their epitope by immunoblotting, we describe the expression of the 2 sarcomeric actins in mature skeletal muscle and during muscle repair after micro-lesions. In particular, we analyze the expression of α-CAA, α-SKA and α-SMA by co-immunostaining in a time course frame during the muscle repair process. Our results indicate that a restricted myocyte population expresses α-CAA and suggest a high capacity of self-renewal in muscle cells. These antibodies may represent a helpful tool for the follow-up of muscle regeneration and pathological changes.",
"keywords": [
"Actin isoforms",
"monoclonal antibodies",
"epitope",
"muscle repair"
],
"content": "Introduction\n\nExpression of actin isoforms in skeletal muscle at the mRNA and protein levels have been described since the 1980s using tissue extracts (Gunning et al., 1983; Hayward & Schwartz, 1986; Hayward et al., 1988; Minty et al., 1982; Ordahl, 1986; Paterson & Eldridge, 1984; Sassoon et al., 1988; Vandekerckhove et al., 1986). These studies have indicated that sarcomeric actins (α-CAA and α-SKA) are expressed in fetal and regenerating skeletal muscle, whereas α-SKA becomes the predominant actin isoform in mature skeletal muscle. Noteworthy, during fetal life, another actin isoform, α-SMA, is highly expressed and precedes the sarcomeric isoforms (Bochaton-Piallat et al., 1992; Hayward & Schwartz, 1986; McHugh et al., 1991; Woodcock-Mitchell et al., 1988).\n\nNevertheless, very little is known concerning the precise localization of these isoforms due to the lack of specific α-SKA and α-CAA antibodies. The first mAb against α-CAA has allowed the identification, by immunohistochemistry, of a α-CAA transient expression in human skeletal muscle satellite cells during skeletal regeneration induced by muscle injury, while normal skeletal muscle was negative (Franke et al., 1996). Ten years later, the same group, in a more extensive study, has further analyzed the expression and localization of α-CAA in normal, regenerating, diseased and neoplastic human muscle tissues (Moll et al., 2006). In human fetal skeletal muscle, a uniform strong α-CAA staining was observed while in normal adult skeletal muscle, α-CAA was identified only in few thin fibers. α-CAA staining of thin fibers became stronger in human regenerating skeletal muscle after traumatic injury, as well as in Duchenne muscular dystrophy (DMD). Whether the persistence of α-CAA in DMD myofibers is due to a lack of differentiation or to a regenerative process deserves further examination. Interestingly, resting satellite cells in healthy adult muscle lack this isoform, whereas, when satellite cells are activated during the regeneration process, α-CAA is up-regulated. Unfortunately, in these two studies, a comparative α-SKA staining was not performed.\n\nIn a more recent study, the switch of α-CAA to α-SKA during the differentiation of skeletal muscle from mouse embryonic stem cells has been examined (Mizuno et al., 2009). In this in vitro system, α-CAA appeared first in myoblasts, with no staining for α-SKA. During cell fusion, α-SKA appeared. When myotubes began to form sarcomers, α-SKA expression increased while α-CAA began to decrease. Finally, mature skeletal muscle fibers were mainly composed of α-SKA. Although, this in vitro system seems to recapitulate the in vivo skeletal muscle differentiation at the level of sarcomeric actins switching, the in vivo origin of progenitor cells during in vivo muscle differentiation/repair remains elusive.\n\nThe sequential expression of the two striated actins during: i) heart development (high expression of α-SKA at birth, predominant expression of α-CAA in differentiated cardiac muscle at adult life) and ii) skeletal muscle development (high expression of α-CAA at birth, predominant of α-SKA in differentiated skeletal muscle at adult life) has been known from mRNA studies for decades, but little is known about the distribution and/or the localization of the two α-sarcomeric muscle actins, because of the lack of double immunostainings availability. Nevertheless, our laboratory, in collaboration with others, has studied α-SKA expression and distribution, in particular in developing and pathological hearts, using affinity polyclonal antibodies (Clément et al., 1999), again without double immunostaining.\n\nAs mentioned above, α-CAA distribution has been studied during muscle development and repair (Moll et al., 2006), but a comparative study with α-SKA was still missing. Our mAbs were raised using the acetylated N-terminus decapeptide of each isoform (see Table 1). The two different mAb subtypes (anti-α-SKA, IgG2b and anti-α-CAA, IgG1), allowed a clear analysis of the expression and distribution of the two actin isoforms in mature skeletal muscle and during regeneration after micro-lesions by means of highly specific anti- mouse subtype secondary antibodies.\n\nThe epitope recognized by each antibody is enlightened in bold.\n\n\nMaterials and methods\n\nDetails of all reagents with reference to the immunoblot and immunostaining procedure can be found in Table 2 and Table 3. Crucial are the conditions of fixation and permeabilization for relevant immunostaining. With cells in culture as well as with tissues, we have tested a large number of conditions such as MeOH, EtOH, PFA-TX100. By far, the use of PFA, followed by MeOH, as described in Table 3 and previously defined (Dugina et al., 2009), allowed the best detection of every actin isoform, likely because of availability of the actin molecule N-terminus.\n\nAll animal experiments (production of antibody in mice, rat wounds, tissue samplings) were approved by and performed in accordance with the cantonal and federal veterinary authorities.\n\nTibialis muscle specimens from female Wistar rats older than 10 weeks were rapidly embedded in OCT compound (Tissue-Tek), snap-frozen in a beaker containing precooled liquid isopentane, immerged in liquid nitrogen, and stored at -80°C. For muscle repair studies, rat tibialis muscles were injured with a liquid nitrogen cooled needle, using a well-set simple rat model (Rocheteau et al., 2012). Muscles were taken, OCT embedded and frozen at different days after injury.\n\nMAbs against α-SKA and α-CAA were prepared following the Repetitive Immunizations Multiple Sites (RIMMS) strategy (Kilpatrick et al., 1997). This strategy uses lymphocytes from regional draining lymph nodes and an immunization schedule significantly shorter than conventional techniques: two weeks instead of several months. Along with the use of less antigen, this approach allows to obtain hybridomas in a month.\n\nMice were immunized with the acetylated N-terminal decapeptide of α-SKA (Ac-DEDETTALVC-COOH) or α-CAA (Ac-DDEETTALVC-COOH) conjugated with keyhole limpet haemocyanin through the cysteine peptide C-terminus (KLH, Imject Maleimide Activated carrier proteins, Pierce) according to the instructions of the manufacturer. Briefly, over a period of 10 days, 5 injections of 5 μg of protein (α-SKA or α-CAA peptide x KLH) emulsified in complete Freund’s adjuvant (first injection) or with incomplete Freund’s adjuvant (for the remaining injections) and RIBI adjuvants (Sigma-Aldrich) were given at six subcutaneous sites proximal to draining peripheral lymph nodes (PLNs) in three anesthetized six-week-old female BALB/c mice (200μl/mice). Two days after the final boosts, animals were sacrificed. PLNs were harvested from popliteal, superficial inguinal, axillary, and brachial lymph nodes and dissociated. Up to 1 × 108 lymphocytes per 3 mice, were used for fusions with 2.5 × 107 with NSO myeloma cells using 50% polyethylene glycol (PEG 1500, Sigma-Aldrich). Fused cells were plated in 24-well tissue culture plates (4000 cells/well). Hybridomas were grown in DMEM+ pyruvate, 5% FCS, 10% NCTC 109 (Sigma-Aldrich), 1× MEM Non-Essential Amino Acids (Gibco), 1% Hybridoma Fusion and Cloning Supplement (HFCS, Roche), penicillin/streptomycin, and 1× selective media hypoxanthine/aminopterin/thymidine (HAT). Supernatants were harvested after 2 weeks and screened for antibody specificity by ELISA and immunofluorescence staining (see below). Hybridomas from wells of interest were distributed into 96-well plates (1-2-5-10 cells/well) for the first limited dilution using 1× hypoxanthine and thymidine (HT) in place of HAT. After about 10 days of culture, wells with single clones were identified by microscope and their supernatants were harvested for screening. Positive clones were re-plated using another limited dilution for a further 10 days in DMEM containing 10% FCS. Clones secreting the specific mAb were expanded and frozen.\n\nSupernatants of hybridoma cells secreting anti-α-SKA or anti-α-CAA were screened by: i) triple ELISA, using 96 well plates coated with α-SMA, α-SKA and α-CAA BSA-conjugated peptides, using Maleimide Activated BSA (Pierce) according to the instructions of the manufacturer; ii) Western blotting using platelets, heart, aorta, skeletal muscle, gizzard extracts (Driesen et al., 2009); iii) immunofluorescence using rat lip, skeletal and heart sections. Selected hybridomas were cloned twice by limited dilution, as described above, and the final mAB characterization was performed by immunoblotting after transfer of one-dimensional gels containing tissue and cell extracts and finally by blocking assays (Chaponnier et al., 1995) using the N-terminal peptides of α-SKA and α-CAA.\n\nPurified α-SKA (Spudich & Watt, 1971) and α-CAA (Zot & Potter, 1981) were run on 10% SDS-PAGE (Laemmli, 1970) and electroblotted to nitrocellulose according to Towbin et al. (Towbin et al., 1979). After preincubation with the respective different length peptides (listed in Figure 1), the antibodies, diluted in Tris-buffered saline (TBS) solution containing 3% BSA and 0.1% Triton X-100, were incubated on membranes strips for two hours at room temperature. After 3 washes with TBS, a second incubation was performed with peroxidase-conjugated affinity purified goat anti-rabbit IgG (Biorad) at a dilution of 1:10,000 in TBS containing 0.1% BSA and 0.1% Triton X-100. Peroxidase activity was developed using the ECL Western blotting system (GE Healthcare), exposed to AX Konica Minolta films for 5–60 sec, and processed with Curix-60 developing machine (Agfa). Blots were scanned and quantified using densitometric analysis ImageJ v1.49 software (NIH, http://rsb.info.nih.gov/ij/).\n\nPurified α-SKA (A) or α-CAA (B) was run on 10% SDS-PAGE and transferred on nitrocellulose membrane. Membrane strips were incubated with the mAb anti-α-SKA (A) or anti-α-CAA (B) alone (control, lane 1) or mixed with the listed peptides (2–9). The epitope recognized by anti-α-SKA (A) includes the acetyl group and the first 7 amino acids of the α-SKA sequence. The epitope recognized by anti-α-CAA (B) includes the acetyl group and the first 5 amino acids of the α-CAA sequence.\n\nCryopreserved tissues were sliced (3 μm) with a cryostat microtome (Microm). Sections were positionned on glass slides, fixed with 1% PFA for 30 min at room temperature, followed by three washes with PBS and a 3 min treatment with methanol at -20°C. After three 5 min washes with PBS at RT, tissue sections on glass slides were incubated with primary (1h) and secondary antibodies (30 min) at appropriate dilutions (see Table 3). Normal rat serum (1:50) was used to block non-specific sites, and DAPI for nuclear staining. After washing in PBS, sections on slides were mounted in polyvinyl alcohol (PVA: 50 mM Tris-phosphate pH 9.0, 0.1% chlorobutanol, 20% polyvinyl alcohol, 0.5‰ phenol red, 20% glycerol) (Lennette, 1978). Images were acquired using a Zeiss Axiophot microscope (Carl Zeiss), equipped with plan apochromatic 10x, 20x, or 40x objectives and a high sensibility color camera (Axiocam, Zeiss).\n\nAfter a careful selection of highly specific secondary antibodies against mouse subtypes, testing a large panel of these antibodies, we selected those commercialized by Jackson Immunoresearch and Southern Biotechnology.\n\n\nResults\n\nAcetylated N-terminal peptide of different length (4–10 amino acids) and non-acetylated N-terminal decapeptide of α-SKA and α-CAA were tested for their blocking ability of the respective mAb (Figure 1). We identified the epitope of α-SKA (AcDEDETTA) and of α-CAA (AcDDEET). The epitope of the other actin isoforms were previously identified and are listed in Table 1 (in bold letters). Noteworthy, the acetyl group is a critical element of the epitope of each isoform.\n\nThe normal myonuclear turnover in rodents is estimated at 1–2% per week (Schmalbruch & Lewis, 2000). As a first investigation, we have compared the distribution of α-CAA with α-SKA, α-SMA and vimentin on cryostat sections of adult rat tibialis muscle. Although a minority of myocytes expressed α-CAA, we have observed two types of α-CAA positive cells in skeletal fibers: one was mainly located in interstitial connective tissue (Figure 2A, a–c, d–f) and the other was localized in the muscle mass (Figure 2A, g–i, arrowhead). The first type corresponded most likely the “muscle spindles”, described by Moll et al. (Moll et al., 2006), as “modified muscle fibers of neuromuscular spindles, believed to act as sensors of muscle tension”. The “spindle” cells expressed exclusively α-CAA, whereas the “classical muscle fibers”, in addition to a high expression of α-CAA, displayed α-SKA at various levels (Figure 2A, g–i, j–l), probably according to the state of differentiation during self-renewal of myocytes.\n\nA) Co-staining with anti-α-SKA (red) and anti-α-CAA (green) allows the detection of isolated thin α-CAA positive fibers in interstitial connective tissue (transversal sections, a–c: longitudinal section, d–f) and of renewing muscle fibers expressing both isoforms (longitudinal section, g–l, arrowhead), or only α-CAA (arrow). Merged images are shown on right column. Bars = 50 μm. B) Co-staining with anti-α-CAA (green) and anti-α-SMA (red) shows that α-CAA positive spindle cells are in close connection with α-SMA positive vessel (a–c), that early muscle fiber self-renewal is characterized by co-expression of both isoform (d–f, arrowhead), although more advanced regenerated fibers are only α-CAA positive (g–i). Merged images are shown on right column. Bar = 50 μm. C) Co-staining with anti-α-CAA (red) and anti-vimentin (green) allows the detection of α-CAA positive muscle spindle cells surrounded by a capsule containing vimentin positive cells (transversal section, a–c) and of renewing α-CAA positive fibers in contact with vimentin positive cells (longitudinal section, d–f). Merged images are shown on right column. Bar = 50 μm.\n\nIt is well known that during skeletal muscle development, α-SMA is the first muscle actin to be expressed in myocytes during fetal life. Therefore, we also investigated the expression of α-SMA during muscle self-renewal. We observed that a few regenerating α-CAA positive fibers displayed α-SMA (Figure 2B, d–f).\n\nAs vimentin is expressed in the capsule of muscle spindle cells (Cizkova et al., 2009), we investigated further whether the α-CAA positive fibers located in interstitial spaces could be identified as component of muscle spindles. After co-staining of muscle sections for α-CAA and vimentin, we confirmed that a capsule containing vimentin-positive cells surrounded isolated α-CAA positive spindle fibers (Figure 2C, a–c). Noteworthy, renovating fibers displayed contacts with vimentin positive cells (Figure 2C, d–f).\n\nThe use of specific mAbs against actin isoforms allows the tracking and follow-up of myocytes renewal during the muscle repair process. In particular, complete and fast repair can be observed after muscle micro-lesions obtained after light injury induced by nitrogen-cooled needle application. At early stage (4 day post-injury), an important population of α-CAA positive myofibers was observed (Figure 3A) at injury sites. These cells, being α-SKA negative or marginally positive, are probably in an early stage of differentiation. Few of them co-expressed α-SMA (Figure 3C, a–c, arrowhead). Only rarely, α-SMA positive myofibroblasts, the hallmark of fibrotic process (Tomasek et al., 2002) were detected (Figure 3C, a–c, arrow). At 5d post-injury, fibers started to express more importantly α-SKA in addition to α-CAA in the regenerating location (Figure 3B, a–f). In our model, muscle regeneration was very rapid, as expression of α-SMA was not any longer detectable 5d after injury (Figure 3C, d–f). At 9d post-injury, co-expression of α-SKA and α-CAA became rare (Figure 3B, g–i). These results suggest that skeletal muscle has a high capacity of regeneration after micro-injury and that actin isoform specific mAbs represent an important tool for muscle regeneration tracking.\n\nA) At 4d post-injury, co-staining with anti-α-SKA (red) and anti-α-CAA (green) shows the presence of α-CAA positive fibers in the injured muscle area (a–i). At this stage of regeneration, only a few fibers co-express both isoforms (d–i), with a low α-SKA level (g–i). Merged images are shown on right column. Bars = 50 μm. B) At 5d–9d post-injury, co-staining with anti-α-SKA (red) and anti-α-CAA (green) shows that after 5d, most fibers co-express both isoforms (a–f), whereas at 9d (g–i), α-SKA positive fibers become predominant. Merged images are shown on right column. Bars = 50 μm. C) At 4d–5d post-injury, co-staining with anti-α-SMA (red) and anti-α-CAA (green) shows that during the healing process, only a few fibers co-express both isoforms after 4d (a–c, arrowhead), whereas after 5d, only α-CAA positive fibers are detected (d–f). After 4d, myofibroblasts might participate to the repair process and are detected by using the anti-α- SMA mAb (a–c, arrow). Merged images are shown on right column. Bars = 50 μm.\n\n\nConclusion\n\nIn conclusion, α-CAA, in conjunction with the expression of α-SMA and α-SKA, appears to represent a valuable marker for the identification of myofibers renewal in skeletal muscle and for the analysis of the degree of fiber differentiation. For this purpose, it is important to use well-characterized and specific antibodies. Furthermore, high quality anti-mouse subtype secondary antibodies allow double immunostaining necessary for this type of investigation.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for Figures 1a, b., 10.5256/f1000research.8154.d117164 (Chaponnier & Gabbiani, 2016).",
"appendix": "Author contributions\n\n\n\nC.C. designed and performed research. C.C. and G.G. wrote the paper.\n\n\nCompeting interests\n\n\n\nThe authors sell the anti-actin isoform antibodies to companies through Unitec, University of Geneva, unitec@unige.ch\n\n\nGrant information\n\nThis work was supported by the Swiss National Science Foundation (grant number 310030_125320) to C.C.\n\n\nAcknowledgments\n\nWe thank Anita Hiltbrunner and Aman Ahmed Mohamed, for their high quality technical assistance, Dr. Marie-Luce Bochaton-Piallat for advice and help with Photoshop and Dr. Stéphane König, Department of Basic Neurosciences, for advice and discussion about the muscle micro-lesion model.\n\n\nReferences\n\nArnoldi R, Hiltbrunner A, Dugina V, et al.: Smooth muscle actin isoforms: a tug of war between contraction and compliance. Eur J Cell Biol. 2013; 92(6–7): 187–200. 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PubMed Abstract | Publisher Full Text\n\nFranke WW, Stehr S, Stumpp S, et al.: Specific immunohistochemical detection of cardiac/fetal alpha-actin in human cardiomyocytes and regenerating skeletal muscle cells. Differentiation. 1996; 60(4): 245–50. PubMed Abstract | Publisher Full Text\n\nGunning P, Ponte P, Blau H, et al.: alpha-skeletal and alpha-cardiac actin genes are coexpressed in adult human skeletal muscle and heart. Mol Cell Biol. 1983; 3(11): 1985–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHayward LJ, Schwartz RJ: Sequential expression of chicken actin genes during myogenesis. J Cell Biol. 1986; 102(4): 1485–93. PubMed Abstract | Free Full Text\n\nHayward LJ, Zhu YY, Schwartz RJ: Cellular localization of muscle and nonmuscle actin mRNAs in chicken primary myogenic cultures: the induction of alpha-skeletal actin mRNA is regulated independently of alpha-cardiac actin gene expression. J Cell Biol. 1988; 106(6): 2077–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKilpatrick KE, Wring SA, Walker DH, et al.: Rapid development of affinity matured monoclonal antibodies using RIMMS. Hybridoma. 1997; 16(4): 381–389. PubMed Abstract | Publisher Full Text\n\nLaemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259): 680–685. PubMed Abstract | Publisher Full Text\n\nLennette DA: An improved mounting medium for immunofluorescence microscopy. Am J Clin Pathol. 1978; 69(6): 647–8. PubMed Abstract\n\nMcHugh KM, Crawford K, Lessard JL: A comprehensive analysis of the developmental and tissue-specific expression of the isoactin multigene family in the rat. Dev Biol. 1991; 148(2): 442–58. PubMed Abstract | Publisher Full Text\n\nMinty AJ, Alonso S, Caravatti M, et al.: A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA. Cell. 1982; 30(1): 185–92. PubMed Abstract | Publisher Full Text\n\nMizuno Y, Suzuki M, Nakagawa H, et al.: Switching of actin isoforms in skeletal muscle differentiation using mouse ES cells. Histochem Cell Biol. 2009; 132(6): 669–72. PubMed Abstract | Publisher Full Text\n\nMoll R, Holzhausen HJ, Mennel HD, et al.: The cardiac isoform of alpha-actin in regenerating and atrophic skeletal muscle, myopathies and rhabdomyomatous tumors: an immunohistochemical study using monoclonal antibodies. Virchows Arch. 2006; 449(2): 175–91. PubMed Abstract | Publisher Full Text\n\nOrdahl CP: The skeletal and cardiac alpha-actin genes are coexpressed in early embryonic striated muscle. Dev Biol. 1986; 117(2): 488–92. PubMed Abstract | Publisher Full Text\n\nPaterson BM, Eldridge JD: alpha-Cardiac actin is the major sarcomeric isoform expressed in embryonic avian skeletal muscle. Science. 1984; 224(4656): 1436–8. PubMed Abstract | Publisher Full Text\n\nRocheteau P, Gayraud-Morel B, Siegl-Cachedenier I, et al.: A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division. Cell. 2012; 148(1–2): 112–25. PubMed Abstract | Publisher Full Text\n\nSassoon DA, Garner I, Buckingham M: Transcripts of alpha-cardiac and alpha-skeletal actins are early markers for myogenesis in the mouse embryo. Development. 1988; 104(1): 155–64. PubMed Abstract\n\nSchmalbruch H, Lewis DM: Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles. Muscle Nerve. 2000; 23(4): 617–26. PubMed Abstract | Publisher Full Text\n\nSkalli O, Ropraz P, Trzeciak A, et al.: A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol. 1986; 103(6 Pt 2): 2787–96. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpudich JA, Watt S: The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971; 246(15): 4866–71. PubMed Abstract\n\nTomasek JJ, Gabbiani G, Hinz B, et al.: Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol. 2002; 3(5): 349–63. PubMed Abstract | Publisher Full Text\n\nTowbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979; 76(9): 4350–4354. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVandekerckhove J, Bugaisky G, Buckingham M: Simultaneous expression of skeletal muscle and heart actin proteins in various striated muscle tissues and cells. A quantitative determination of the two actin isoforms. J Biol Chem. 1986; 261(4): 1838–43. PubMed Abstract\n\nWoodcock-Mitchell J, Mitchell JJ, Low RB, et al.: Alpha-smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles. Differentiation. 1988; 39(3): 161–6. PubMed Abstract | Publisher Full Text\n\nZot HG, Potter JD: Purification of actin from cardiac muscle. Prep Biochem. 1981; 11(4): 381–95. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13281",
"date": "08 Apr 2016",
"name": "Naohiro Hashimoto",
"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\nHigher vertebrates express six different highly conserved actin isoforms. The authors determined the precise localization of alpha-skeletal actin (aSKA) and alpha-cardiac actin (aCAA) in normal and regenerating rat skeletal muscles. The distinct distribution of the two alpha actin isoforms seems very interesting. However, the authors should describe the results of their immunofluorescent analysis on skeletal muscle more precisely and carefully. The authors often used terms “renewing muscle (fibers)” and “regenerating fibers”. However, there is no definition of them. The authors should show the presence of centrally located nuclei in those myofibers because those are found in newly formed myofibers. The authors identified myofibroblasts in regenerating skeletal muscles as the alpha-SMA (aSMA) positive mononuclear cells. However, myoblasts also express aSMA. Thus, the authors should determine the absence of myogenic lineage markers such as MyoD in those cells. Intracellular localization of alpha actin isoforms should be shown in pictures with higher magnification in addition to the present Figure 2 and 3. Those data will provide important information suggesting different function of those actin isoforms. The term “self-renewal” is used incorrectly. For examples, “self-renewal in muscle cells” and “during muscle self-renewal” are difficult to understand. I wonder whether “muscle cells” mean muscle satellite cells, myoblasts (muscle progenitor cells), or myofibers.° “Muscle self-renewal” means muscle regeneration?",
"responses": [
{
"c_id": "1994",
"date": "01 Jun 2016",
"name": "Christine Chaponnier",
"role": "Author Response",
"response": "Your careful review and suggestions for improving the report are highly appreciated. Please find below our answers to your questions. a) We have modified the text according to the suggestion using in general the term regeneration and not renewal. b) Enlargement of Figure 2Af and 3Ai showing αCAA positive myofibers with centrally located nuclei during muscle regeneration. (See supplemental Figure 2) a) The injury model used in this study is in general not accompanied by fibrotic events during the healing process. Only, in rare exceptions, as mentioned in the text, myofibroblasts were detected. We have a long experience in the “myofibroblast history”, and according to the α-SMA positivity and the cell shape, we believe that this area is composed of myofibroblasts. b) We agree that the absence of MyoD would validate our assumption. However, the purpose of this report is the validation of the actin isoforms antibodies and not a extensive study on muscle regeneration. We hope that muscle experts will take advantage of these tools for more advanced studies. As indicated above, the purpose of this report is the validation of the actin isoforms antibodies. The intracellular localization of α-SMA, sometimes in striations, will not give clear information concerning its function in muscle regeneration. a) See answer to point 1 b) Future studies by experts, hopefully using the panel of antibodies against actin isoforms, will provide more information for the participation of satellite cells during the process of muscle regeneration."
}
]
},
{
"id": "13310",
"date": "11 Apr 2016",
"name": "Omar Skalli",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Chaponnier and Gabbiani reports the production of two monoclonal antibodies, one specific for skeletal actin and the other for cardiac actin. Being able to obtain these antibodies is a momentous accomplishment given that these two actin isoforms differ from each other by only a few amino acids. The authors rigorously demonstrate the specificity of each antibodies for their respective isoform by Western blotting and competitive assay with the immunogen. Because the antibodies are from different subtypes, they can be used by double immunofluorescence to investigate the regulation of skeletal and cardiac actin in different conditions. The authors provide stunningly beautiful immunofluorescence images that demonstrate the utility of their antibodies to pinpoint different cellular populations during skeletal muscle repair. It is to be expected that these antibodies will be extremely useful to studies questions related to cardiac and skeletal muscle physiology and pathology.",
"responses": [
{
"c_id": "1993",
"date": "01 Jun 2016",
"name": "Christine Chaponnier",
"role": "Author Response",
"response": "Your nice review and perfect understanding concerning the objectives of this report are highly appreciated."
}
]
},
{
"id": "13217",
"date": "12 Apr 2016",
"name": "Shoichiro Ono",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDrs. Chaponnier and Gabbiani report production and validation of monoclonal antibodies that specifically recognize alpha-skeletal muscle actin (aSKA) or alpha-cardiac alpha actin (aCAA), which are different only in a few amino acids at the N-terminus. The new antibody against aSKA (10D2) is IgG2a, while anti-aCAA (22D3) is IgG1, which allows researchers to perform double staining in immunohistochemistry to distinguish these closely related actin isoforms. The validation in immunohistochemistry using rat tissues clearly demonstrates specific labeling of different subsets of cells by the isoform-specific antibodies. These antibodies should be outstanding resources to characterize actin isoform expression and subcellular localization in muscle development, regeneration, and diseases, as well as to investigate functions of actin isoforms. I have a few suggestions for a revision mostly for clarification purposes.In abstract and introduction, “higher vertebrates” should be more clearly defined. Classification of six actin isoforms in 3 subgroups applies to mammals and birds. It should be useful for researchers who use non-mammalian or non-avian species to state whether this classification applies to other vertebrates. A recent analysis by Gunning et al.1 indicates that zebrafish has 10 muscle actins, suggesting some gene duplications and isoform divergence in fish. Page 3, left column, 3rd paragraph, “sarcomers” should be “sarcomeres”. In Materials and Methods “Antibody production and details”, this section should also include description of how the monoclonal antibodies were prepared after the clonal selection has been completed, which should include whether the antibodies were finally prepared in culture media with or without serum or in ascites, and whether protein concentrations were determined. Also In Materials and Methods “Antibody production and details”, the selection process includes screening using Western blotting using extracts from several different tissues. To improve transparency of the validation process, representative images of the Western blots preferably showing a wide molecular weight range should be included to demonstrate specificity.",
"responses": [
{
"c_id": "1992",
"date": "01 Jun 2016",
"name": "Christine Chaponnier",
"role": "Author Response",
"response": "Your careful review and suggestions for improving the report are highly appreciated. Please find below our answers to your questions. a) We have modified the abstract accordingly. b) Concerning the amino acid sequences in zebrafish, the situation is very confusing. Search in databases gives the following results: Actin, alpha 2, smooth muscle, aorta Danio rerio (Zebrafish) (Brachydanio rerio) Q6DHS1-1 Ac-DDEESTALVC Actin, alpha cardiac muscle 1 [Danio rerio] Q6IQR3-1 Ac-DDDETTALVC Actin, alpha 1, skeletal muscle Danio rerio (Zebrafish) (Brachydanio rerio) Q9I8V1-1 Ac-DDEETTALVC According to the available sequence (although not reviewed), the N-terminus sequence of alpha 1, skeletal muscle in Zebrafish is identical to the alpha cardiac actin in mammals and birds!!! Furthermore, the N-terminus of alpha 2, smooth muscle, aorta and of alpha cardiac muscle are different compared to the α-SMA and α-CAA human and bird sequence. sarcomers changed for sarcomeres. Additional informations have been included in the \"Material and Methods\" section. Yes the final antibody is obtained and used in “hybridoma supernatant” in the culture media with FCS for two reasons: a) Ascites production is not allowed in Switzerland for years. b) Hybridoma supernatants production is anyway privileged, as ascites are very often contaminated with unrelated antibodies with production time. c) Protein concentration was not determined precisely (in general, between 0.1-10 μg/ml). However, the dilution to be used is indicated in the report. We provide a new figure (Supplemental figure 1), with raw data of Western blots on different tissue extracts, showing the specificity of the antibodies."
}
]
}
] | 1
|
https://f1000research.com/articles/5-416
|
https://f1000research.com/articles/5-1048/v1
|
31 May 16
|
{
"type": "Opinion Article",
"title": "Teaching and learning based on peer review: a realistic approach in forensic sciences",
"authors": [
"Ricardo Jorge Dinis-Oliveira",
"Teresa Magalhães",
"Teresa Magalhães"
],
"abstract": "Teaching and learning methods need a continuous upgrade in higher education. However it is also true that some of the modern methodologies do not reduce or prevent school failure. Perhaps the real limitation is the inability to identify the true reasons that may explain it or ignore/undervalue the problem. In our opinion, one of the current constraints of the teaching/learning process is the excess of and inadequate bibliography recommended by the teacher, which results in continuous student difficulties and waste of time in searching and selecting useful information. The need to change the paradigm of the teaching/learning process comes also from employers. They claim forensic experts armed with useful knowledge to face professional life. It is therefore mandatory to identify the new needs and opportunities regarding pedagogical methodologies. This article reflects on the recent importance of peer review in teaching/learning forensic sciences based on the last 10 years of pedagogical experience inseparably from the scientific activity.",
"keywords": [
"peer-review",
"teaching",
"learning",
"forensic sciences"
],
"content": "Introduction\n\nTeaching and communicating with students is a very personal experience, shaped by the idiosyncrasy of each teacher but also dependent on the individual and motivational characteristics of the students as well as the institutional environment and the methodologies and pedagogical tools used by the teacher. Challenges of today’s society, particularly of economic, social and scientific nature, and the mass access to continuous new information, urges a revolution in the way of thinking on education. It is needed to renew the paradigm of teaching and it should be made every effort to ensure that the university and the professional life are seen as partners in research and education1.\n\nThe model of teaching/learning based on the Bologna Process2 designed to ensure comparability in the standards and quality of higher education qualifications, emphasizes the development of skills such as critical thinking, and attributes to the student an active role in his/her own training. It is obvious that these components are desirable and worth exploring, but there is still a long way to go. Resulting perhaps of a misunderstanding, the Bologna Process was sometimes assumed as an educational unaccountability of the teacher’s functions, transposing to the student almost the entire workload of the learning process. The challenge for higher education claims a continuous and systematic teaching/learning in order to build up capacities to define and solve problems, search for and select relevant scientific information, formulate hypotheses in an interdisciplinary basis, clearly communicate the results and draw conclusions. Indeed, learning is an ongoing process that occurs throughout life and is not limited and does not end in the syllabus of any curricular unit (academic discipline). Indeed, “once a student, always a student”. It is not only crucial, but also urgent aiming at reaching higher academic success rates that teachers assure detailed curricular plans, well-adjusted to the objectives, avoiding the huge amount of student’s workload almost impossible to accomplish. This is obvious regarding the frequent oversized bibliography recommendation.\n\nIn a system that favors the qualities developed and displayed in research, teaching is a field left to free will of each teacher and without proper investment and scrutiny by peers. It is for many teachers the poor relation of their work and the most often overlooked part. If the scientific component is subject to peer review, why do not take this reality also in teaching/learning process?\n\nThe purpose of this article is to reflect on the importance of the scientific peer review in teaching/learning forensic sciences and its inherent potential in offering a more reasonable and realistic education. This reflection is based on our teaching experiences and on our student’s feedback.\n\n\nTeaching methodologies\n\nSince the last half of the twentieth century, most of the teaching methodologies, especially those applied in theoretical classes, were based on the transmission of information through transparency acetates, followed by slides and nowadays supported by datashow presentations. Teachers commonly assume that this type of documentation is the recommended study material, though not always prepared with the best quality and amount of information. Additionally, a non-criterions bibliography is recommended suggesting an “unaccountability” of the teacher of his/her functions. Indeed, is this bibliography of adequate size (often based on multiple sources not easily accessible to students) to the real available time for study/personal work? Do students effectively consult it, or on the other hand stay limited to the inefficiency of datashows presentations? Does the teacher have the right to request/recommend hundred pages after a single contact hour? Were the teachers able to achieve this transmission level in a packed auditorium with the continuous increase of the numerus clausus? Of course it is desirable and recommended that students develop routines for seeking information, but it is also important some dose of realism.\n\n\nCurricular syllabus and recommended bibliography\n\nThe development of the syllabus of any curricular unit is a privileged moment for reflection, particularly on the permanent adaptation of content and teaching methods to constant social changes of community life. It is also an excellent time for a look at our own journey as teachers in a permanent dissatisfaction behavior. Frequently, one of the great student difficulties is the immensity of bibliographic resources that can be used for study purposes. Although being excessive, recommended bibliography is insufficient to reverse the school failure. It is the teacher’s obligation to adjust and be realistic/reasonable when making available resources and contents. The exigence, rigor and seriousness of education is not measured by the number of pages that are recommended for study. At this level, the disrespect for those we teach transcends even the most skilled.\n\nNowadays, teaching Forensic Sciences acquires great importance in several academic areas such as medicine, pharmaceutical sciences, chemistry, biology, etc.3. It refers broadly to the use of science in matters of law4. It is used to identify a growing number of specialties and subspecialties, particularly in medicine and life sciences, that recurs to scientifically valid and legally admissible methods to clarify evidence that is being examined, or may be in the future, at judicial and judiciary level5, under criminal or some other branch of the law. There are therefore separate disciplines within the Forensic Sciences6. Noteworthy is that due to the multidisciplinary nature of forensic work and the way by which these sciences are organized in each country, it is not always easy to have useful bibliography for teaching Forensic Sciences. It was exactly this literature gap that prompted adjustments in the teaching/learning process of Forensic Sciences, leading to introducing peer review in classes preparation.\n\n\nPeer review in higher education\n\nIn the case of scientific literature, the peer review is an assessment of the soundness of the theme, originality and interest to the scientific community as well as the adequacy and accuracy of the methodology, results, discussion and conclusions, and also the relevance of citations. Although generally it does not ensure the veracity of content, peer review undoubtedly increases the quality of most scientific articles7,8.\n\nBased on the importance that this peer has acquired in recent years, we have been particularly attentive and committed to use scientific scrutiny as a pedagogical opportunity to teach Forensic Sciences in the 1st, 2nd and 3rd Cycle of Studies, specialization and other continuing education courses that we coordinate3. For this reason, several classes were written in a review/didactic article format, always aiming to adjust the recommended bibliography to what is actually taught in the classroom. These articles were then submitted for publication and are currently already available for students particularly in Forensic Toxicology. To accomplish this process it was crucial to respect the binomial education/research.\n\nUnlike the requirements inherent to the most common datashow presentations, writing a scientific paper requires an additional effort in terms of time and reflection, which allows a good systematization of information and selection of literature. It produces consequently a non-comparable study document in terms of quality and usefulness. We believe that this educational pathway, besides being an effort of humility and scientific honesty aiming at the transmission of sound and useful knowledge, is also important to make students aware of the need of focusing their medical practice on scientific evidence and not just on empiric routines and mere opinions. Of course, writing classes in this format represents an immense team effort, but it is also rewarding. Although we have not, yet, a quantitative evaluation of academic success in pre- and post-publication scenario, much due to the fact that peer review is often a slow, sometimes expensive, subjective and prone to bias, there is no doubt that we have more satisfied students (including those already graduated and therefore more exigent), who effectively consult bibliography in the form of a scientific paper or book (and not limited to datashow presentations). It is also assured that students assimilate knowledge of useful quantity and quality and teachers are much more confident since the scrutiny of the peer review guaranties fewer errors and greater introspection regarding the topics focused in class. This type of teaching methodology represents also an effort of educating students aiming to transform the “campus in a laboratory”9.\n\n\nFinal remarks\n\nTeaching and learning Forensic Sciences is based on a solid foundation of theoretical and/or practical knowledge promoting correct expert practices. It is important that the transmitted skills are based on proven scientific knowledge not only in autodidactic practices that leads over the years into vices and inevitable erroneous judicial decisions. Our practices for teaching and learning embodies some breaks with the past. One of them is the need for continuous evaluation of teachers by peer review, in order to improve students’ academic results.\n\nAs mentioned above, teaching and learning are ongoing processes and should promote the development of general and specific competences (in a particular professional field), and structure basic knowledge. In the age of entrepreneurship, realism is for us the philosophy of education. Therefore, it is legitimate to aim to a greater interconnection between academia and industry and offer students the knowledge of their practical life. To achieve this goal, it was truly useful and rewarding, to send classes in article format for peer review, in addition to the scrutiny carried out by the students through educational surveys. Pedagogical plurality and learning with peers are crucial aspects to better teach. In fact, this type of continuous teacher’s evaluation can represent an additional catalysis of learning10. In full agreement with a recent Nature editorial11, universities cannot continue with teaching methodologies based on the idea that only the most capable students will survive; we need to learn how to build a professor and researcher of the XXI century to have students with relevant competences. It is expected that peer review can be considered as a parameter for increasing the quality of classes in higher education.",
"appendix": "Author contributions\n\n\n\nDinis-Oliveira RJ prepared the first draft of the manuscript.\n\nDinis-Oliveira RJ and Magalhães T discussed the content and wrote the final version. Both authors assume the full responsibility for the article and agreed with the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nDinis-Oliveira RJ acknowledges Fundação para a Ciência e a Tecnologia (FCT) for his Investigator Grant (IF/01147/2013).\n\n\nReferences\n\nWatson-Capps JJ, Cech TR: Academia and industry: Companies on campus. Nature. 2014; 514(7522): 297–298. PubMed Abstract | Publisher Full Text\n\nScott P: Going Beyond Bologna: Issues and Themes. In: Curaj A, Scott P, Vlasceanu L, Wilson L, eds. European Higher Education at the Crossroads: Between the Bologna Process and National Reforms. Springer, Netherlands, Dordrecht, 2012; 1–14. Publisher Full Text\n\nMagalhães T, Dinis-Oliveira RJ, Santos A: Teaching forensic medicine in the University of Porto. J Forensic Leg Med. 2014; 25: 45–48. PubMed Abstract | Publisher Full Text\n\nCrispino F, Houck MM: Principles of forensic science. In: Siegel JA, Saukko PJ, eds. Encyclopedia of forensic sciences. Academic Press, Waltham, 2013; 278–281. Publisher Full Text\n\nWecht CH: The history of legal medicine. J Am Acad Psychiatry Law. 2005; 33(2): 245–251. PubMed Abstract\n\nDinis-Oliveira RJ, Magalhães T: What are forensic sciences? Concepts, scope and future perspectives. Lisbon Lidel-Edições Técnicas; 2016. Reference Source\n\nDinis-Oliveria RJ, Magalhães T: The Inherent Drawbacks of the Pressure to Publish in Health Sciences: Good or Bad Science [version 1; referees: 2 approved]. F1000Res. 2015; 4: 419. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith R: Opening up BMJ peer review. BMJ. 1999; 318(7175): 4–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe university experiment: Campus as laboratory. Nature. 2014; 514(7522): 288–291. PubMed Abstract | Publisher Full Text\n\nBoud D, Cohen R, Sampson J: Peer learning and assessment. Assess Eval High Educ. 1999; 24(4): 413–426. Publisher Full Text\n\nSTEM education: To build a scientist. Nature. 2015; 523: 371–373. Publisher Full Text"
}
|
[
{
"id": "14499",
"date": "21 Jun 2016",
"name": "Hugo Filipe Violante Cardoso",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reflection provided in this article is scientifically solid and sound. The abstract summarizes well the contents of the published work and, although the title is appropriate, it carries some ambiguity about what exactly the review process refers to. This would probably require a rather long and inconvenient title and, therefore, the current one seems appropriate. The paper is of a reflective nature, but outlines clearly the problem, the particular solution that was undertaken in an attempt to solve it, and some of the results. It would have been interesting to read more details about the results of the peer-review process itself and the actual impact in the classroom, but this is perhaps a first attempt at materializing some of the authors’ thoughts and experience that will be developed further in other publications or reports. The conclusion consolidates the authors’ opinion that peer-review as a pedagogical tool enhances the connection between scientifically generated knowledge and forensic practice in a higher education setting. As an article that focuses on the teachers’ wishes to improve on lecture delivery quality and teachers’ pedagogic accountability, it successfully outlines a classroom approach that has much to offer. I am certain that many teachers will be looking forward to knowing more about the learning outcomes of this approach and will give it strong consideration as a pedagogical tool.",
"responses": []
},
{
"id": "15085",
"date": "04 Aug 2016",
"name": "Mauricio Yonamine",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nForensic science needs a multidisciplinary approach and requires knowledge from different areas such as sciences of life and death, natural sciences, technology and law from students. In fact, its complexity demands new pedagogical methodologies, in view that conventional classes based on datashow presentations and complementary studies in multiple bibliography sources do not seem to be effective in the learning process. Based on this context, the discussion raised by the authors is completely valid and their arguments seem to be logical. Certainly this teaching strategy is much more laborious, however, it can generate contemporary knowledge for both professors and students, providing the opportunity to receive other expert´s opinions on the covered topic. We believe that the authors could enrich the discussion if more results about the teaching methodology were presented, including the student’s opinion, which would enhance the manuscript and strengthen this practice. In addition, the process of teaching and learning could also be improved with the presence of professionals in the area, as forensic experts. Their experience acquired in cases resolution could be taken to the classroom, which would be an opportunity for students to apply the acquired theoretical knowledge in real forensic cases. Students could discuss about the proposed problem, presenting their hypothesis and revealing their understanding. In the end, the case resolution could also be presented in the form of a forensic case report and submitted to the peer review process. By doing so, the subject would present theoretical and practical strenght.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1048
|
https://f1000research.com/articles/5-1046/v1
|
31 May 16
|
{
"type": "Review",
"title": "Recent advances in understanding idiopathic pulmonary fibrosis",
"authors": [
"Cécile Daccord",
"Toby M. Maher",
"Cécile Daccord"
],
"abstract": "Despite major research efforts leading to the recent approval of pirfenidone and nintedanib, the dismal prognosis of idiopathic pulmonary fibrosis (IPF) remains unchanged. The elaboration of international diagnostic criteria and disease stratification models based on clinical, physiological, radiological, and histopathological features has improved the accuracy of IPF diagnosis and prediction of mortality risk. Nevertheless, given the marked heterogeneity in clinical phenotype and the considerable overlap of IPF with other fibrotic interstitial lung diseases (ILDs), about 10% of cases of pulmonary fibrosis remain unclassifiable. Moreover, currently available tools fail to detect early IPF, predict the highly variable course of the disease, and assess response to antifibrotic drugs.\n\nRecent advances in understanding the multiple interrelated pathogenic pathways underlying IPF have identified various molecular phenotypes resulting from complex interactions among genetic, epigenetic, transcriptional, post-transcriptional, metabolic, and environmental factors. These different disease endotypes appear to confer variable susceptibility to the condition, differing risks of rapid progression, and, possibly, altered responses to therapy. The development and validation of diagnostic and prognostic biomarkers are necessary to enable a more precise and earlier diagnosis of IPF and to improve prediction of future disease behaviour. The availability of approved antifibrotic therapies together with potential new drugs currently under evaluation also highlights the need for biomarkers able to predict and assess treatment responsiveness, thereby allowing individualised treatment based on risk of progression and drug response. This approach of disease stratification and personalised medicine is already used in the routine management of many cancers and provides a potential road map for guiding clinical care in IPF.",
"keywords": [
"idiopathic pulmonary fibrosis",
"interstitial lung diseases"
],
"content": "Introduction\n\nIdiopathic pulmonary fibrosis (IPF) is typically introduced as a chronic progressive and inevitably fatal scarring lung disease with a prognosis worse than that of numerous cancers1,2. Hopefully, this is now beginning to change. Although the etiology and the pathogenesis of IPF are still incompletely understood, two antifibrotic drugs, pirfenidone and nintedanib, have recently been proven to be effective in slowing disease progression and are now approved as treatments in the United States and Europe3,4.\n\nThe recent development of affordable, high-throughput -omics technologies has opened the era of systems biology and has enabled the emergence of stratified and personalised medicine. These approaches are becoming routine practice in oncology5 and have enormous potential in offering new insights into the understanding and management of pulmonary diseases6, including IPF.\n\nThis article aims to provide an overview of recent developments in disentangling the complex interrelated mechanisms involved in the pathogenesis of IPF with a particular focus on those that may lead to improved diagnosis, stratification of disease behaviour, and identification of potential novel therapeutic targets and predictors of response to treatment. Considerations concerning the past, present, and future pharmacotherapy of IPF were addressed in the March 2014 issue of this journal7 and will not be discussed in this current review.\n\n\nDiagnosis\n\nThe current approach to IPF diagnosis was first described in international guidelines published in 2001, which were recently updated. These guidelines define precise diagnostic criteria based on clinical, radiological, and histopathological features8 and enshrine the place of multidisciplinary discussion among experienced clinicians, radiologists, and pathologists as the gold standard method for establishing a diagnosis of IPF. Using the current guidelines, in about two-thirds of the cases, a confident diagnosis of IPF can be achieved based on an appropriate clinical history in association with a typical high-resolution computed tomography (HRCT) pattern of usual interstitial pneumonia (UIP) (Figure 1). When clinical and HRCT data are non-diagnostic, surgical lung biopsy (SLB) is recommended to confirm UIP diagnosis histologically (Figure 2). However, SLB carries considerable risks and is often contraindicated in older patients with extensive co-morbidities or in those presenting with advanced lung disease9. Thus, even in experienced centres, a diagnosis of unclassifiable interstitial lung disease (ILD) is assigned to about 10% of patients who present with progressive pulmonary fibrosis10.\n\nThe image shows subpleural and basal predominance of reticular opacities associated with traction bronchiectasis and honeycomb change (clustered cystic airspaces with well-defined thick walls and diameter of 0.3–1.0 cm).\n\nThe patient shows the typical histopathological features of usual interstitial pneumonia characterised by spatial heterogeneity with areas of subpleural and paraseptal fibrosis and honeycombing changes (cystic airspaces lined by bronchiolar epithelium) alternating with areas of relatively spared lung parenchyma, temporal heterogeneity with admixed areas of active fibrosis with fibroblast foci, extracellular matrix deposition (mainly collagen), and relative mild or absence of inflammatory cell infiltrate together with regions of histologically normal lung tissue.\n\nIn a recent study of 117 patients with fibrotic ILDs, bronchoscopic lung cryobiopsy has proven to be safe and effective in providing adequate lung tissue samples, which enabled increased diagnostic confidence in the multidisciplinary diagnosis of IPF11. This minimally invasive technique represents an attractive alternative to SLB and may, pending further studies, be included in the diagnostic algorithm of IPF and other fibrotic ILDs in the near future.\n\n\nPathogenesis\n\nThe heterogeneity in radiological and histopathological appearances, rate of progression, and treatment response observed in individuals with IPF suggests that fibrosis arises as a consequence of multiple co-activated pathogenic pathways, all of which are influenced by complex interactions between endogenous and environmental factors12. This multiple-pathway model probably explains the disappointing results of therapies targeting single receptors or pathways in IPF. Future treatment strategies in IPF are likely to focus on combinations of therapies targeting multiple pathogenic pathways simultaneously, as is currently used in the treatment of many cancers13.\n\nUntil 15 years ago, the prevailing pathogenic paradigm in IPF was one of chronic inflammation being the precursor to progressive fibrosis. This has shifted over the last decade to a model of abnormal wound healing response driven by persistent or recurrent alveolar epithelial microinjuries (e.g. cigarette smoke, microaspiration, or infection) in individuals rendered susceptible by ageing or genetic predisposition14. Multiple studies have shown that alveolar epithelial cell (AEC) apoptosis secondary to injury is followed by extravascular coagulation, immune system activation, and aberrant persistent activation of AECs, even in the absence of the primary stimulus15. These cells, in turn, induce the migration and proliferation of local fibroblasts, recruit circulating fibrocytes to areas of injury, and promote differentiation of fibroblasts into myofibroblasts. This results in the formation of myofibroblast foci, the histologic hallmark of UIP, in which persistently activated myofibroblasts secrete excessive amounts of extracellular matrix (ECM) proteins. Disordered deposition and accumulation of ECM components within the interstitium and alveolar spaces lead to established fibrosis with progressive destruction of lung architecture and loss of function.\n\nThis pathogenic cascade involves complex cell-cell and cell-matrix interactions through numerous biochemical mediators, such as growth factors, enzymes, chemokines, coagulation factors, and reactive oxygen species, all of which have the potential to be influenced by numerous host and environmental factors16–19. Cardinal among these is transforming growth factor-beta (TGF-β), a potent profibrotic mediator involved in cell recruitment, myofibroblast differentiation, and induction of ECM production18–19 (Figure 3).\n\nIn genetically susceptible individuals, injury activates multiple inflammatory, cell signalling, and repair pathways. Activation of these cascades causes an imbalance in profibrotic and antifibrotic mediators. In turn, these mediators activate multiple cell types, causing changes in cellular functioning and cell-cell interactions that ultimately result in progressive fibrosis. Abbreviations: CTGF, connective tissue growth factor; FXa, factor Xa; HGF, hepatocyte growth factor; IFNγ, interferon-γ; PDGF, platelet-derived growth factor; PGE2, prostaglandin E2; TGFβ, transforming growth factor β, Th, T-helper; VEGF, vascular endothelial growth factor.\n\n\nDisease stratification and personalised medicine\n\nThe early manifestations of IPF are, in the absence of a biopsy, frequently difficult to distinguish from other ILDs. Furthermore, the histological hallmark of IPF, UIP, is found in other disorders and so even when a biopsy is available a diagnosis of IPF can remain in doubt. A further challenge for clinicians is the fact that currently available clinical measures do not allow accurate prediction of subsequent disease behaviour that can range from slowly to rapidly progressive and that, in 5% of cases, is punctuated by episodes of rapid acute deterioration or acute exacerbation20.\n\nThese challenges highlight the need for the development and validation of diagnostic markers specific to IPF and prognostic markers of future disease behaviour to guide treatment decisions, including referral for transplant21. The recent approval of pirfenidone and nintedanib and the identification of new potential therapeutic targets have created an urgent need for theragnostic markers, i.e. markers able to assess, ideally at an early stage, therapeutic response to a given drug. Such markers could be used to improve patient selection in clinical trials and also to personalise treatment based on an individual’s risk of progression and treatment response. This in turn would avoid unnecessarily exposing individuals to side effects and would improve the cost-effectiveness of treatment. This approach of disease stratification and personalised medicine is already used in the routine management of cancers and has the potential to improve clinical care in IPF.\n\n\nClinical phenotyping\n\nSeveral clinical, physiologic, radiographic, and pathologic variables enable a certain degree of mortality prediction in IPF. Older age, male sex, smoking history, low body mass index (BMI), pulmonary hypertension, and concomitant emphysema are clinical predictors of worse survival20. Longitudinal changes in forced vital capacity (FVC) and diffusion capacity for carbon monoxide (DLCO) are more predictive of prognosis than baseline values. Thus, a 5–10% decline in FVC at 6 months is associated with a more than twofold increase in the risk of mortality over the subsequent year22. Using relative change in FVC instead of the absolute change enables earlier detection of progression with similar prognostic accuracy23. Also reported as independent predictors of mortality are baseline 6-minute walk distance (6MWD) and change in 6MWD at 6 months24.\n\nAdditionally, several multi-dimensional risk prediction models integrating various clinical, physiological, and radiological variables have been validated in IPF25–29 (Table 1). These composite staging systems are more accurate in predicting baseline and longitudinal mortality risk than individual physiological variables and permit stratification of IPF patients into groups with distinct patterns of survival. Nevertheless, they cannot reliably predict future disease behaviour (as measured by rate of decline in FVC) or response to treatment30. Additionally, they provide no insights into underlying pathobiology and thus fail to identify distinct molecular phenotypes of disease. The integration of dynamic parameters measured over time and biological biomarkers able to reflect disease activity is needed to improve the accuracy of disease stratification models and guide personalised management31.\n\nAbbreviations: DLCO, diffusing capacity of carbon monoxide; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 second; CT, computed tomography; % pred, % predicted; pts, points.\n\n*GAP calculator for more precise estimation of risk available at www.annals.org\n\nInterestingly, the development and greater accessibility of 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) may provide a novel method for evaluating disease activity in IPF. Areas of established honeycomb fibrosis appear to be highly metabolically active, as shown by increased 18F-FDG uptake on PET/CT32. More importantly, increased 18F-FDG uptake is also observed in areas of radiologically normal lung parenchyma on HRCT, suggesting that PET/CT may have a higher sensitivity than HRCT in detecting early disease in IPF and may thus represent a potential useful tool in monitoring disease activity and response to treatment33, albeit one which is limited by radiation exposure.\n\n\nMolecular phenotyping\n\nHigh-throughput -omics technologies enable the rapid, accurate, and simultaneous analysis of high numbers of genes, RNA transcripts, proteins, or metabolites. This in turn has facilitated the emergence of systems biology, a multidisciplinary methodology based on integration models aimed at understanding biological systems as a whole, i.e. as a dynamic network of complex interrelated networks extending from the genome to the environment. This contrasts with linear models that have been used in the past to explain the action of individual genes and proteins6. Such multi-scale modelling should permit mapping of the considerable phenotypic heterogeneity of IPF and may enable the identification of specific molecular phenotypes associated with clinical outcomes that could be used to improve diagnosis accuracy and disease stratification21 (Table 2).\n\nAbbreviations: AE, acute exacerbation; BALF, bronchoalveolar lavage fluid; cCK18, caspase-cleaved cytokeratin-18; CCL18, CC-chemokine ligand 18; CXCL13, C-X-C motif chemokine 13; DKC1, dyskeratosis congenital 1 or dyskerin; ECM, extracellular matrix; HSP, heat shock protein; ICAM-1, intercellular adhesion molecule-1; IL-8, interleukin-8; ILDs, interstitial lung diseases; KL-6/MUC1, Krebs von den Lungen-6/Mucin 1; LOXL2, lysyl oxidase-like 2; LYCAT, lysocardiolipin acyltransferase; miRNAs, microRNAs; MMP, matrix metalloproteinases; MUC5B, mucin 5B; PH, pulmonary hypertension; SFTPA2, surfactant protein A2 gene; SFTPC, surfactant protein C gene; RTEL1, regulator of telomere elongation helicase 1; SNPs, single nucleotide polymorphisms; TERC, telomerase RNA component; TERT, telomerase reverse transcriptase; TOLLIP, Toll-interactive protein; Tregs, regulatory T cells.\n\nIdeal molecular biomarkers should reflect key pathological pathways, be easily and accurately measured, have been validated, and offer added value to currently used approaches34. IPF stratification and personalised management based on molecular biomarkers is not yet available in current clinical practice, but recent advances in understanding the complex pathobiology of IPF has identified candidate biomarkers involved in AEC dysfunction, immune dysregulation, ECM remodelling, and fibroproliferation35. A prerequisite for the use of biomarkers in clinical practice is validation in large well-phenotyped cohorts with longitudinal follow up of both clinical and molecular parameters. Among several cohort studies are the COMET (Correlating Outcomes With Biochemical Markers to Estimate Time to progression in Idiopathic Pulmonary Fibrosis) study in the United States36–38 and the PROFILE (Prospective Observation of Fibrosis in the Lung Clinical Endpoints) study in the United Kingdom39,40. The latter is the largest prospective cohort study of incident IPF with over 550 patients recruited, all of whom were naïve for antifibrotic therapy at the time of inclusion.\n\n\nGenetic phenotyping\n\nTwo large genome-wide association studies (GWAS) have identified several common genetic variants associated with susceptibility to IPF and risk of disease progression. The genes identified are involved in host defence, cell-cell adhesion, and DNA repair41,42. A single nucleotide polymorphism (SNP) in the promoter region of the MUC5B gene, encoding a mucin involved in airway host defence43, is significantly associated with sporadic and familial IPF44 and, paradoxically, with improved survival45. This MUC5B promoter polymorphism is not associated with lung fibrosis in scleroderma or sarcoidosis and thus appears to be specific to IPF46. Similarly, several SNPs conferring susceptibility to IPF have been identified within the TOLLIP locus42. The TOLLIP gene encodes for a protein with reduced expression in patients with IPF and that regulates part of the innate immune system mediated by Toll-like receptor and TGF-β signalling pathways. Surprisingly, the minor allele rs5743890 in TOLLIP appears to be protective against the development of IPF but when present tends to be associated with increased mortality.\n\nStudies based on familial IPF have identified rare genetic variants in genes encoding surfactant proteins, including surfactant protein C (SFTPC) and A2 (SFTPA2), and in several genes linked to telomere function, such as TERT (which encodes for telomerase reverse transcriptase, a component of the telomerase complex responsible for maintaining telomere length47). Short telomere length as well as evidence of lung parenchymal remodelling and epithelial dysfunction have been identified in asymptomatic first-degree relatives of familial IPF patients and may represent the earliest stages of IPF48. Even in the absence of TERT polymorphisms, short telomeres in peripheral blood mononuclear cells (PBMCs) or in AECs are also frequently found in IPF patients and portend a poorer prognosis47,49. This suggests that both genetic variants and environmental factors such as cigarette smoke play a role in telomere shortening.\n\nThe biological role of the various genetic variants in the pathogenesis of IPF has yet to be fully determined. Interestingly, an exploratory post hoc study conducted in a subgroup of patients participating in a multi-centre randomised control trial of N-acetylcysteine treatment for IPF suggests that genetic polymorphisms may play a role in determining N-acetylcysteine treatment response50. This remains to be confirmed in a prospective clinical trial.\n\n\nTranscriptional phenotyping\n\nWhole RNA microarray analysis of lung tissue from patients with different ILDs has identified disease-specific gene expression signatures that permit UIP to be identified from non-UIP samples51,52. Furthermore, the comparison of lung gene expression profiles of patients with stable or rapidly progressive IPF has identified 134 transcripts sufficiently upregulated or downregulated in the progressive IPF group to distinguish stable from progressive disease53. Similarly, analysis of the peripheral blood transcriptome in IPF has identified genes differentially expressed between IPF patients and healthy controls and also between those with mild and severe disease54,55. For example, mRNA expression of lysocardiolipin acyltransferase (LYCAT), a cardiolipin-remodelling enzyme, in PBMCs of IPF patients appeared to be strongly correlated with lung function parameters and survival56.\n\nThe identification of these diagnostic or prognostic gene expression signatures is a first step towards the development of molecular tests that could be applied to bronchoscopy samples or peripheral blood, thus allowing less invasive approaches to the diagnosis of IPF and earlier identification of individuals at risk of rapid progression.\n\n\nEpigenetic and microRNA regulation phenotyping\n\nDNA methylation57,58, histone modifications59,60, and noncoding microRNAs (miRNAs)61 are epigenetic mechanisms identified as contributing to differences in gene expression observed in IPF. These regulatory mechanisms are influenced by various factors including environmental exposures (cigarette smoke and infection), genetic profile, sex, and ageing62. A genome-wide DNA methylation analysis of lung tissue identified 2130 significantly differentially methylated regions in IPF samples compared to controls, of which about a third were associated with significant changes in gene expression, including genes identified as IPF-associated common genetic variants63. Thus, dysregulated gene expression in the IPF lung appears to result from complex interactions between genetic and epigenetic factors.\n\nmiRNAs influence protein expression by binding to mRNA. Aberrant expression of miRNAs has been described in the pathogenesis of many cancers. Lung tissue miRNA profiling identified significantly increased64 or decreased65 levels of several regulatory miRNAs in IPF patients, thereby distinguishing the normal lung from the IPF lung and rapidly progressive from slowly progressive disease66. TGF-β seems to play a critical role in the upregulation of profibrotic miRNAs and downregulation of antifibrotic miRNAs67. For example, the direct inhibition of let-7d expression by TGF-β in AECs is associated with epithelial to mesenchymal transition and collagen deposition68. Similarly, several circulating miRNAs appear to be differentially expressed in the serum of IPF patients67. Moreover, the expression levels of miR-21, miR-155, and miR-101-3p in serum seem to be correlated with FVC and HRCT features of IPF69. Interestingly, in mice, intravenous injection of synthetic miR-29 during bleomycin-induced pulmonary fibrosis restored endogenous miR-29 function and was followed by decreasing collagen expression and reversal of pulmonary fibrosis70. These changes in miRNA expression in IPF patients suggest that they play an important regulatory role in lung fibrosis and may represent potential diagnostic and prognostic biomarkers as well as therapeutic targets.\n\n\nProtein and cell biomarkers\n\nA growing number of studies have sought to identify protein- and cell-based predictors of IPF disease behaviour. Elevated serum levels of several proteins have been associated with worse prognosis in IPF, including surfactant protein A (SP-A) and D (SP-D)71,72, mucin 1 (KL-6/MUC1)73, CC-chemokine ligand 18 (CCL18)74, C-X-C motif chemokine 13 (CXCL13)75,76, periostin77, fibulin-178, matrix metalloproteinases MMP-1 and MMP-779,80, interleukin-8 (IL-8), intercellular adhesion molecule (ICAM)-180, and lysyl oxidase-like 2 protein (LOXL2)81. Elevated baseline serum levels of KL-6/MUC1 also appear to predict the risk of future acute exacerbation82. Similarly, some circulating cells have been associated with worse survival. Among cellular markers of rapidly progressive IPF are elevated circulating fibrocytes83 and semaphorin 7a+ regulatory T cells (Tregs)84.\n\nSerial measurements of serum ECM protein fragments generated by MMP activity in 189 IPF patients recruited in the PROFILE cohort identified increased serum concentrations of these protein fragments in IPF patients compared to controls. More importantly, increasing neoepitope concentrations were associated with disease progression, and the rate of change over 3 months of 3 of these MMP-degraded ECM proteins predicted survival40. These results suggest that serial longitudinal measurement of circulating proteins have potential for use as prognostic or theragnostic biomarkers.\n\nStudies based on lung tissue or bronchoalveolar lavage fluid (BALF) analysis have also identified some candidate diagnostic and prognostic biomarkers of IPF, including αvβ6 integrin85, S100A9 protein86, and soluble annexin V87.\n\nThe value of these protein or cell biomarkers as diagnostic or prognostic factors in IPF needs to be further assessed. Furthermore, integrating validated molecular variables in multivariate risk prediction models could improve their accuracy in predicting outcomes in IPF. In view of this, Richards and colleagues formulated the personal clinical and molecular index (PCMI), integrating sex, FVC % predicted, DLCO % predicted, and MMP-7 serum concentration, which accurately predicted mortality in their validation cohort80. Two other prediction models integrating SP-A and SP-D levels or MMP-7, SP-A, and KL-6/MUC1 levels have shown improved predictability of mortality compared with clinical predictors alone71,88.\n\n\nMetabolic phenotyping\n\nMetabolomics is the systematic analysis of the complete set of metabolites (the metabolome) within a biological system under given conditions. This approach offers the potential for a better understanding of dysregulated metabolic pathways underlying numerous diseases, including airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis89. Dysregulated metabolic mechanisms have also been highlighted in the pathogenesis of IPF. Increased levels of lactic acid in IPF lung tissue compared with controls appear to play a role in myofibroblast differentiation via a pH-dependent activation of TGF-β90. Recently, a metabolomic assay by Xie and colleagues demonstrated that augmented aerobic glycolysis, mediated by upregulated glycolytic enzymes, including PFKFB3, represented an early and sustained event during myofibroblast differentiation91. More importantly, PFKFB3 inhibition mitigated myofibroblast differentiation and dampened the profibrotic phenotypes of myofibroblasts isolated from IPF lungs. These data suggest that glycolytic reprogramming is important in the pathogenesis of lung fibrosis and therefore represents a potential therapeutic target. More research is needed in the field of metabolomics to clarify the role of these dysregulated pathways of cellular metabolism in the pathogenesis of IPF and to integrate them with available genetic, epigenetic, transcriptomic, and proteomic data.\n\n\nEnvironmental and host factors\n\nSmoking history has long been described as a prevalent risk factor for the development of IPF92, including familial IPF93, and is associated with a worse survival94. Some other environmental and occupational exposures, including wood, mineral, and metal dusts, agriculture, and livestock, have also been associated with IPF, although a formal causal link has not been established95. Furthermore, air pollution may also play a role in the pathogenesis of IPF. A recent study reported a significantly higher risk of acute exacerbation of IPF with increased ozone and nitrogen dioxide exposure over the preceding 6 weeks96.\n\nGastroesophageal reflux (GER) is highly prevalent in IPF, though often asymptomatic, and confers an increased risk of microaspiration97. Anti-acid treatment in IPF has been associated in retrospective data with decreased radiologic fibrosis, longer survival, and smaller decrease of FVC at 30 weeks98,99. Despite growing evidence suggesting that GER and silent microaspiration might play a role in the pathogenesis of IPF, there is, to date, no confirmation that this association is causative. Consequently, the recently updated international guidelines on IPF treatment maintained a conditional recommendation for the use of anti-acid therapy100. A prospective randomised controlled trial is needed to further assess the role of GER and microaspiration in IPF and confirm the effectiveness of anti-reflux therapy.\n\nInfectious processes may play a role in the initiation, progression, or exacerbation of IPF. Viral infections, particularly human herpes viruses (HHVs), including herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), cytomegalovirus (CMV), HHV-7, and HHV-8, have been associated with IPF in several studies101. Whether this association is causative has not yet been proven. HHVs have the potential to induce endoplasmic reticulum stress and apoptosis102; it is therefore hypothesised that viral infection may act as a cofactor in the development of IPF through the reactivation of latent HHVs within the alveolar epithelium following exposure to a first injury103. Furthermore, a recent study found increased copy numbers of EBV and CMV DNA in BALF of IPF patients and, to a lesser extent, in first-degree asymptomatic relatives of familial IPF patients48. Thus, enhanced HHV replication may trigger epithelial cell stress and participate in disease initiation. A small clinical trial of ganciclovir in individuals with severe IPF with positive EBV-IgG serology showed a modest improvement in surrogate markers of disease progression104. It has recently been reported that influenza infection may also play a role in lung fibrosis by promoting collagen deposition via αvβ6 integrin-mediated TGF-β activation in epithelial cells105.\n\nRecent data also suggest a putative role for bacteria and lung microbiome in IPF. An analysis of the COMET study showed an association between progression of IPF and the presence of specific members within the Staphylococcus and Streptococcus genera in BALF38. Similarly, Molyneaux and colleagues found an increased bacterial load, consisting particularly of Haemophilus, Streptococcus, Neisseria, and Veillonella spp., in BALF of IPF patients compared to healthy smokers, nonsmokers, and patients with moderate COPD106. More importantly, the total bacterial burden was an independent predictor of decline in lung function and mortality. Whether these differences in lung microbiome are a cause or consequence of IPF is unknown. A clinical trial of 12 months of co-trimoxazole in addition to standard treatment in 181 patients with fibrotic idiopathic interstitial pneumonia (about 90% of whom had IPF) showed a reduction in mortality but did not slow functional decline107. The exact role of viruses and bacteria in the pathogenesis of IPF has yet to be determined and the potential for antiviral or antibiotic treatments requires further evaluation.\n\n\nConclusion\n\nCurrently available therapies for IPF are of limited efficacy, and the prognosis associated with the condition remains poor. Recent advances in our understanding of the complex interrelated mechanisms underlying fibrosis in the lung are encouraging and pave the way towards an integrated approach to diagnosis, stratification, and treatment. It is becoming increasingly clear that genetic polymorphisms, whole blood transcriptomic profile, and lavage microbiome all predict groups of patients with differing disease behaviour and outcomes and potentially variable responses to treatment. Furthermore, prospective longitudinal cohort studies have started to identify blood biomarkers that have the potential to be used as early measures of treatment response. Considerable further research is required to deliver personalised medicine for IPF into the clinic, but at least now there is light at the end of what has been a very long tunnel.",
"appendix": "Competing interests\n\n\n\nCécile Daccord does not have competing interests to report. Toby Maher has no declarations directly related to this manuscript. He has, however, received industry-academic research funding from GlaxoSmithKline R&D, UCB, and Novartis and has received consultancy or speakers fees from Apellis, Bayer, Biogen Idec, Boehringer Ingelheim, Dosa, GlaxoSmithKline R&D, Galapagos, Novartis, ProMetic, Roche, Sanofi-Aventis, and UCB.\n\n\nGrant information\n\nCécile Daccord is supported by Ligue Pulmonaire Vaudoise, Service de pneumologie CHUV, and Fonds de perfectionnement CHUV. Toby M Maher is supported by an NIHR Clinician Scientist Fellowship (NIHR Ref: CS-2013-13-017) and receives additional infrastructure support from the Royal Brompton NIHR funded biomedical research unit.\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\nHutchinson J, Fogarty A, Hubbard R, et al.: Global incidence and mortality of idiopathic pulmonary fibrosis: a systematic review. Eur Respir J. 2015; 46(3): 795–806. PubMed Abstract | Publisher Full Text\n\nVancheri C, Failla M, Crimi N, et al.: Idiopathic pulmonary fibrosis: a disease with similarities and links to cancer biology. Eur Respir J. 2010; 35(3): 496–504. PubMed Abstract | Publisher Full Text\n\nKing TE Jr, Bradford WZ, Castro-Bernardini S, et al.: A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis. N Engl J Med. 2014; 370(22): 2083–92. 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PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWells AU, Desai SR, Rubens MB, et al.: Idiopathic pulmonary fibrosis: a composite physiologic index derived from disease extent observed by computed tomography. Am J Respir Crit Care Med. 2003; 167(7): 962–9. PubMed Abstract | Publisher Full Text\n\ndu Bois RM, Weycker D, Albera C, et al.: Ascertainment of individual risk of mortality for patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2011; 184(4): 459–66. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLey B, Ryerson CJ, Vittinghoff E, et al.: A multidimensional index and staging system for idiopathic pulmonary fibrosis. Ann Intern Med. 2012; 156(10): 684–91. PubMed Abstract | Publisher Full Text\n\nLey B, Bradford WZ, Weycker D, et al.: Unified baseline and longitudinal mortality prediction in idiopathic pulmonary fibrosis. Eur Respir J. 2015; 45(5): 1374–81. 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PubMed Abstract | Publisher Full Text\n\nWin T, Thomas BA, Lambrou T, et al.: Areas of normal pulmonary parenchyma on HRCT exhibit increased FDG PET signal in IPF patients. Eur J Nucl Med Mol Imaging. 2014; 41(2): 337–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLey B, Brown KK, Collard HR: Molecular biomarkers in idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2014; 307(9): L681–91. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSpagnolo P, Tzouvelekis A, Maher TM: Personalized medicine in idiopathic pulmonary fibrosis: facts and promises. Curr Opin Pulm Med. 2015; 21(5): 470–8. PubMed Abstract | Publisher Full Text\n\nHuie TJ, Moss M, Frankel SK: What can biomarkers tell us about the pathogenesis of acute exacerbations of idiopathic pulmonary fibrosis? Am J Physiol Lung Cell Mol Physiol. 2010; 299(1): L1–2. PubMed Abstract | Publisher Full Text\n\nNaik PK, Bozyk PD, Bentley JK, et al.: Periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2012; 303(12): L1046–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHan MK, Zhou Y, Murray S, et al.: Lung microbiome and disease progression in idiopathic pulmonary fibrosis: an analysis of the COMET study. Lancet Respir Med. 2014; 2(7): 548–56. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMaher TM: PROFILEing idiopathic pulmonary fibrosis: rethinking biomarker discovery. Eur Respir Rev. 2013; 22(128): 148–52. PubMed Abstract | Publisher Full Text\n\nJenkins RG, Simpson JK, Saini G, et al.: Longitudinal change in collagen degradation biomarkers in idiopathic pulmonary fibrosis: an analysis from the prospective, multicentre PROFILE study. Lancet Respir Med. 2015; 3(6): 462–72. PubMed Abstract | Publisher Full Text\n\nFingerlin TE, Murphy E, Zhang W, et al.: Genome-wide association study identifies multiple susceptibility loci for pulmonary fibrosis. Nat Genet. 2013; 45(6): 613–20. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nNoth I, Zhang Y, Ma SF, et al.: Genetic variants associated with idiopathic pulmonary fibrosis susceptibility and mortality: a genome-wide association study. Lancet Respir Med. 2013; 1(4): 309–17. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRoy MG, Livraghi-Butrico A, Fletcher AA, et al.: Muc5b is required for airway defence. Nature. 2014; 505(4783): 412–6. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSeibold MA, Wise AL, Speer MC, et al.: A common MUC5B promoter polymorphism and pulmonary fibrosis. N Engl J Med. 2011; 364(16): 1503–12. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPeljto AL, Zhang Y, Fingerlin TE, et al.: Association between the MUC5B promoter polymorphism and survival in patients with idiopathic pulmonary fibrosis. JAMA. 2013; 309(21): 2232–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nStock CJ, Sato H, Fonseca C, et al.: Mucin 5B promoter polymorphism is associated with idiopathic pulmonary fibrosis but not with development of lung fibrosis in systemic sclerosis or sarcoidosis. Thorax. 2013; 68(5): 436–41. PubMed Abstract | Publisher Full Text\n\nKropski JA, Blackwell TS, Loyd JE: The genetic basis of idiopathic pulmonary fibrosis. Eur Respir J. 2015; 45(6): 1717–27. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKropski JA, Pritchett JM, Zoz DF, et al.: Extensive phenotyping of individuals at risk for familial interstitial pneumonia reveals clues to the pathogenesis of interstitial lung disease. Am J Respir Crit Care Med. 2015; 191(4): 417–26. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nStuart BD, Lee JS, Kozlitina J, et al.: Effect of telomere length on survival in patients with idiopathic pulmonary fibrosis: an observational cohort study with independent validation. Lancet Respir Med. 2014; 2(7): 557–65. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nOldham JM, Ma SF, Martinez FJ, et al.: TOLLIP, MUC5B, and the Response to N-Acetylcysteine among Individuals with Idiopathic Pulmonary Fibrosis. Am J Respir Crit Care Med. 2015; 192(12): 1475–82. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSelman M, Pardo A, Barrera L, et al.: Gene expression profiles distinguish idiopathic pulmonary fibrosis from hypersensitivity pneumonitis. Am J Respir Crit Care Med. 2006; 173(2): 188–98. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKim SY, Diggans J, Pankratz D, et al.: Classification of usual interstitial pneumonia in patients with interstitial lung disease: assessment of a machine learning approach using high-dimensional transcriptional data. Lancet Respir Med. 2015; 3(6): 473–82. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBoon K, Bailey NW, Yang J, et al.: Molecular phenotypes distinguish patients with relatively stable from progressive idiopathic pulmonary fibrosis (IPF). PLoS One. 2009; 4(4): e5134. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nYang IV, Luna LG, Cotter J, et al.: The peripheral blood transcriptome identifies the presence and extent of disease in idiopathic pulmonary fibrosis. PLoS One. 2012; 7(6): e37708. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMeltzer EB, Barry WT, Yang IV, et al.: Familial and sporadic idiopathic pulmonary fibrosis: making the diagnosis from peripheral blood. BMC Genomics. 2014; 15(1): 902. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHuang LS, Mathew B, Li H, et al.: The mitochondrial cardiolipin remodeling enzyme lysocardiolipin acyltransferase is a novel target in pulmonary fibrosis. Am J Respir Crit Care Med. 2014; 189(11): 1402–15. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSanders YY, Ambalavanan N, Halloran B, et al.: Altered DNA methylation profile in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2012; 186(6): 525–35. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHuang SK, Scruggs AM, McEachin RC, et al.: Lung fibroblasts from patients with idiopathic pulmonary fibrosis exhibit genome-wide differences in DNA methylation compared to fibroblasts from nonfibrotic lung. PLoS One. 2014; 9(9): e107055. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCoward WR, Watts K, Feghali-Bostwick CA, et al.: Defective histone acetylation is responsible for the diminished expression of cyclooxygenase 2 in idiopathic pulmonary fibrosis. Mol Cell Biol. 2009; 29(15): 4325–39. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCoward WR, Watts K, Feghali-Bostwick CA, et al.: Repression of IP-10 by interactions between histone deacetylation and hypermethylation in idiopathic pulmonary fibrosis. Mol Cell Biol. 2010; 30(12): 2874–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDakhlallah D, Batte K, Wang Y, et al.: Epigenetic regulation of miR-17~92 contributes to the pathogenesis of pulmonary fibrosis. Am J Respir Crit Care Med. 2013; 187(4): 397–405. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYang IV, Schwartz DA: Epigenetics of idiopathic pulmonary fibrosis. Transl Res. 2015; 165(1): 48–60. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nYang IV, Pedersen BS, Rabinovich E, et al.: Relationship of DNA methylation and gene expression in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2014; 190(11): 1263–72. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMilosevic J, Pandit K, Magister M, et al.: Profibrotic role of miR-154 in pulmonary fibrosis. Am J Respir Cell Mol Biol. 2012; 47(6): 879–87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPandit KV, Corcoran D, Yousef H, et al.: Inhibition and role of let-7d in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2010; 182(2): 220–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOak SR, Murray L, Herath A, et al.: A micro RNA processing defect in rapidly progressing idiopathic pulmonary fibrosis. PLoS One. 2011; 6(6): e21253. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCui H, Xie N, Thannickal VJ, et al.: The code of non-coding RNAs in lung fibrosis. Cell Mol Life Sci. 2015; 72(18): 3507–19. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nYang G, Yang L, Wang W, et al.: Discovery and validation of extracellular/circulating microRNAs during idiopathic pulmonary fibrosis disease progression. Gene. 2015; 562(1): 138–44. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLi P, Li J, Chen T, et al.: Expression analysis of serum microRNAs in idiopathic pulmonary fibrosis. Int J Mol Med. 2014; 33(6): 1554–62. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMontgomery RL, Yu G, Latimer PA, et al.: MicroRNA mimicry blocks pulmonary fibrosis. EMBO Mol Med. 2014; 6(10): 1347–56. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKinder BW, Brown KK, McCormack FX, et al.: Serum surfactant protein-A is a strong predictor of early mortality in idiopathic pulmonary fibrosis. Chest. 2009; 135(6): 1557–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarlo NP, van Moorsel CH, Ruven HJ, et al.: Surfactant protein-D predicts survival in patients with idiopathic pulmonary fibrosis. Sarcoidosis Vasc Diffuse Lung Dis. 2009; 26(2): 155–61. PubMed Abstract\n\nYokoyama A, Kondo K, Nakajima M, et al.: Prognostic value of circulating KL-6 in idiopathic pulmonary fibrosis. Respirology. 2006; 11(2): 164–8. PubMed Abstract | Publisher Full Text\n\nPrasse A, Probst C, Bargagli E, et al.: Serum CC-chemokine ligand 18 concentration predicts outcome in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2009; 179(8): 717–23. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nVuga LJ, Tedrow JR, Pandit KV, et al.: C-X-C motif chemokine 13 (CXCL13) is a prognostic biomarker of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2014; 189(8): 966–74. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDePianto DJ, Chandriani S, Abbas AR, et al.: Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis. Thorax. 2015; 70(1): 48–56. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nNaik PK, Bozyk PD, Bentley JK, et al.: Periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2012; 303(12): L1046–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJaffar J, Unger S, Corte TJ, et al.: Fibulin-1 predicts disease progression in patients with idiopathic pulmonary fibrosis. Chest. 2014; 146(4): 1055–63. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRosas IO, Richards TJ, Konishi K, et al.: MMP1 and MMP7 as potential peripheral blood biomarkers in idiopathic pulmonary fibrosis. PLoS Med. 2008; 5(4): e93. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRichards TJ, Kaminski N, Baribaud F, et al.: Peripheral blood proteins predict mortality in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2012; 185(1): 67–76. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nChien JW, Richards TJ, Gibson KF, et al.: Serum lysyl oxidase-like 2 levels and idiopathic pulmonary fibrosis disease progression. Eur Respir J. 2014; 43(5): 1430–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nOhshimo S, Ishikawa N, Horimasu Y, et al.: Baseline KL-6 predicts increased risk for acute exacerbation of idiopathic pulmonary fibrosis. Respir Med. 2014; 108(7): 1031–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMoeller A, Gilpin SE, Ask K, et al.: Circulating fibrocytes are an indicator of poor prognosis in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2009; 179(7): 588–94. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nReilkoff RA, Peng H, Murray LA, et al.: Semaphorin 7a+ regulatory T cells are associated with progressive idiopathic pulmonary fibrosis and are implicated in transforming growth factor-β1-induced pulmonary fibrosis. Am J Respir Crit Care Med. 2013; 187(2): 180–8. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSaini G, Porte J, Weinreb PH, et al.: αvβ6 integrin may be a potential prognostic biomarker in interstitial lung disease. Eur Respir J. 2015; 46(2): 486–94. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHara A, Sakamoto N, Ishimatsu Y, et al.: S100A9 in BALF is a candidate biomarker of idiopathic pulmonary fibrosis. Respir Med. 2012; 106(4): 571–80. PubMed Abstract | Publisher Full Text\n\nBuckley S, Shi W, Xu W, et al.: Increased alveolar soluble annexin V promotes lung inflammation and fibrosis. Eur Respir J. 2015; 46(5): 1417–29. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSong JW, Do KH, Jang SJ, et al.: Blood biomarkers MMP-7 and SP-A: predictors of outcome in idiopathic pulmonary fibrosis. Chest. 2013; 143(5): 1422–9. PubMed Abstract | Publisher Full Text\n\nNobakht M Gh BF, Aliannejad R, Rezaei-Tavirani M, et al.: The metabolomics of airway diseases, including COPD, asthma and cystic fibrosis. Biomarkers. 2015; 20(1): 5–16. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKottmann RM, Kulkarni AA, Smolnycki KA, et al.: Lactic acid is elevated in idiopathic pulmonary fibrosis and induces myofibroblast differentiation via pH-dependent activation of transforming growth factor-β. Am J Respir Crit Care Med. 2012; 186(8): 740–51. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nXie N, Tan Z, Banerjee S, et al.: Glycolytic Reprogramming in Myofibroblast Differentiation and Lung Fibrosis. Am J Respir Crit Care Med. 2015; 192(12): 1462–74. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBaumgartner KB, Samet JM, Stidley CA, et al.: Cigarette smoking: a risk factor for idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 1997; 155(1): 242–8. PubMed Abstract | Publisher Full Text\n\nSteele MP, Speer MC, Loyd JE, et al.: Clinical and pathologic features of familial interstitial pneumonia. Am J Respir Crit Care Med. 2005; 172(9): 1146–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAntoniou KM, Hansell DM, Rubens MB, et al.: Idiopathic pulmonary fibrosis: outcome in relation to smoking status. Am J Respir Crit Care Med. 2008; 177(2): 190–4. PubMed Abstract | Publisher Full Text\n\nTaskar VS, Coultas DB: Is idiopathic pulmonary fibrosis an environmental disease? Proc Am Thorac Soc. 2006; 3(4): 293–8. PubMed Abstract | Publisher Full Text\n\nJohannson KA, Vittinghoff E, Lee K, et al.: Acute exacerbation of idiopathic pulmonary fibrosis associated with air pollution exposure. Eur Respir J. 2014; 43(4): 1124–31. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSavarino E, Carbone R, Marabotto E, et al.: Gastro-oesophageal reflux and gastric aspiration in idiopathic pulmonary fibrosis patients. Eur Respir J. 2013; 42(5): 1322–31. PubMed Abstract | Publisher Full Text\n\nLee JS, Ryu JH, Elicker BM, et al.: Gastroesophageal reflux therapy is associated with longer survival in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2011; 184(12): 1390–4. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLee JS, Collard HR, Anstrom KJ, et al.: Anti-acid treatment and disease progression in idiopathic pulmonary fibrosis: an analysis of data from three randomised controlled trials. Lancet Respir Med. 2013; 1(5): 369–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRaghu G, Rochwerg B, Zhang Y, et al.: An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline: Treatment of Idiopathic Pulmonary Fibrosis. An Update of the 2011 Clinical Practice Guideline. Am J Respir Crit Care Med. 2015; 192(2): e3–19. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMolyneaux PL, Maher TM: The role of infection in the pathogenesis of idiopathic pulmonary fibrosis. Eur Respir Rev. 2013; 22(129): 376–81. PubMed Abstract | Publisher Full Text\n\nLawson WE, Crossno PF, Polosukhin VV, et al.: Endoplasmic reticulum stress in alveolar epithelial cells is prominent in IPF: association with altered surfactant protein processing and herpesvirus infection. Am J Physiol Lung Cell Mol Physiol. 2008; 294(6): L1119–26. PubMed Abstract | Publisher Full Text\n\nKropski JA, Lawson WE, Blackwell TS: Right place, right time: the evolving role of herpesvirus infection as a \"second hit\" in idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2012; 302(5): L441–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEgan JJ, Adamali HI, Lok SS, et al.: Ganciclovir antiviral therapy in advanced idiopathic pulmonary fibrosis: an open pilot study. Pulm Med. 2011; 2011: 240805. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJolly L, Stavrou A, Vanderstoken G, et al.: Influenza promotes collagen deposition via αvβ6 integrin-mediated transforming growth factor β activation. J Biol Chem. 2014; 289(51): 35246–63. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMolyneaux PL, Cox MJ, Willis-Owen SA, et al.: The role of bacteria in the pathogenesis and progression of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2014; 190(8): 906–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShulgina L, Cahn AP, Chilvers ER, et al.: Treating idiopathic pulmonary fibrosis with the addition of co-trimoxazole: a randomised controlled trial. Thorax. 2013; 68(2): 155–62. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "14068",
"date": "31 May 2016",
"name": "Paul W Noble",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14069",
"date": "31 May 2016",
"name": "Simon P Hart",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1046
|
https://f1000research.com/articles/5-1045/v1
|
31 May 16
|
{
"type": "Review",
"title": "Advancing cardiovascular tissue engineering",
"authors": [
"George A. Truskey"
],
"abstract": "Cardiovascular tissue engineering offers the promise of biologically based repair of injured and damaged blood vessels, valves, and cardiac tissue. Major advances in cardiovascular tissue engineering over the past few years involve improved methods to promote the establishment and differentiation of induced pluripotent stem cells (iPSCs), scaffolds from decellularized tissue that may produce more highly differentiated tissues and advance clinical translation, improved methods to promote vascularization, and novel in vitro microphysiological systems to model normal and diseased tissue function. iPSC technology holds great promise, but robust methods are needed to further promote differentiation. Differentiation can be further enhanced with chemical, electrical, or mechanical stimuli.",
"keywords": [
"Tissue engineering",
"cardiovascular",
"iPSCs"
],
"content": "Introduction\n\nTissue engineering involves the development of functional replacements for damaged tissues or organs (http://www.nibib.nih.gov/science-education/science-topics/tissue-engineering-and-regenerative-medicine). A common approach to produce engineered tissues is to add cells to a natural or synthetic extracellular matrix, which provides mechanical support and biochemical cues. Scaffold-free tissues are prepared by growing cells on thermally responsive polymers to facilitate the cell monolayers that form and then adding layers together or rolling the sheets. By addition of small molecules that activate specific differentiation pathways, three-dimensional organoids can be derived from human pluripotent stem cells1. The engineered tissue may be prepared wholly or partially before implantation to activate and localize the body’s regenerative capacity to populate the implanted scaffold. Tissue engineering is a subset of the broader field of regenerative medicine, which seeks to repair or replace damaged organs. This could occur by direct injection of cells or modifying cellular processes to initiate repair and regrowth. In spite of significant research advances and insightful application of developmental cell biology, cell mechanics, and biomaterials, few products have emerged from these efforts to date, pointing to the challenges to develop truly functional tissues.\n\nKey design goals to produce functional tissues in vitro are to reproduce the tissue structure and cell density in vivo, identify suitable sources of cells, promote growth and differentiation of cells, design a construct that reproduces the extracellular matrix with the appropriate molecular cues and suitable mechanical properties, and create a vasculature within the construct to enable oxygenation and integration with host vasculature after implantation.\n\nSeveral strategies that have emerged to address these challenges include the use of induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) that can differentiate into the cells of interest, the reprogramming of primary cells to the cell type of interest, various ways to engineer the structural support for the cells to mimic the extracellular matrix, and efforts to promote vascular network formation (Figure 1). Over the past few years, a new use of tissue engineering has emerged in which microscale human tissue-engineered systems or microphysiological systems are used to model normal and disease states in vitro and assess drug responses.\n\nPrimary cells or induced pluripotent stem (iPS) cells undergo a period of proliferation prior to seeding into a three-dimensional scaffold or are grown as sheets on a polymer whose conformation changes in response to temperature or other stimuli, enabling detachment on the sheet. During the proliferation phase, microRNAs or transcription factors may be added to the cells to promote subsequent differentiation. To promote further differentiation, small molecules are added and/or cells are exposed to electrical or mechanical stimuli. Endothelial cells added to the tissue during formation promote vascular network formation. After the cells have reached a certain level of maturity, the engineered tissue is implanted and the host blood supply connects with the vascular network promoted by endothelial cells. Alternatively, cells may be encapsulated in a biodegradable polymer and implanted.\n\nRecent advances in tissue engineering over the past three years were recently summarized in two reviews2,3. Given the breadth of tissue engineering research (13,661 publications since 1 January 2014 reported on Google Scholar), we focus this summary of recent work on cardiovascular tissue engineering as a way to demonstrate how new research results have addressed the key design challenges. Cardiovascular tissue engineering is a vibrant area of research, and applications in the cardiovascular system include cardiac patches, engineered blood vessels and heart valves, and vascular networks.\n\n\nScaffolds\n\nScaffold materials should match the mechanical properties of the tissue and undergo degradation or be integrated into the tissue, allowing the natural extracellular matrix to replace the original structural support. Materials used in cardiovascular tissue engineering include degradable polymers, such as polyglycolic acid4 and polylactic acid, as well as biological hydrogels, such as collagen5, fibrin6, and modified hyaluronic acid7. These scaffold materials can be modified by the addition of cell adhesion domains or sites susceptible to cleavage by matrix metalloproteinases to facilitate cell attachment and migration. Alternatively, sheets of cells can be prepared and then fabricated into different configurations such as tubes or patches. Such structures have high mechanical strength and function well in vivo8. This approach has the advantage of not needing any synthetic polymers.\n\nA scaffold-free cardiac patch consisting of three layers of rat cardiomyocytes was successfully engrafted onto heart tissue by overlaying the patch over a vascular supply, enhancing the ability of endothelial cells (ECs) in the patch to form a functional tubular vascular network connected to the host blood supply9. Recently, the development of a porous patch with an electroactive polypyrrole incorporating electronics for sensing and stimulating electrophysiological activity and release of various biological molecules offers a new level of control of cardiac patches while permitting incorporation of cells attached to a bioactive scaffold10.\n\nWhile tissue-engineered blood vessels (TEBVs) constructed from natural matrix components such as collagen11,12 and fibrin13,14 have traditionally exhibited poor mechanical strength, plastic compression of collagen gels embedded with smooth muscle cells (SMCs) increases the collagen fiber density and yields rapidly producible tubular structures with high mechanical strength15. By plastic compression of collagen, TEBVs with burst pressures exceeding 1600 mmHg can be prepared in a few hours5. After one week of perfusion at physiological shear stresses, the medial cells exhibited differentiation and contracted in response to phenylephrine. While these TEBVs have not been studied in vivo, this rapid method of fabrication could significantly reduce the time to produce functional TEBVs.\n\nDecellularized tissue contains many of the cues needed for cells to differentiate and responds dynamically after implantation, owing to cellular infiltration and imposed biomechanical loads. For example, after implantation of decellularized valves in sheep, collagen reorganized, responding to biomechanical stresses16. Increased waviness of collagen corresponded to areas of greater elastin synthesis16. Decellularization does cause loss and damage to some extracellular matrix proteins. To overcome this limitation, the addition of hyaluronic acid supplement enhanced adhesion in decellularized heart tissue17. While decellularized tissue as thick as 1–1.5 cm can be produced, mesenchymal stem cells (MSCs) added to the decellularized constructs reached a cell density of 30 million cells/cm3 but occupied only the outer 100 µm of the decellularized heart, suggesting that their growth was limited by oxygen levels. When the constructs were perfused, cells migrated as far as 400 µm into the decellularized tissue. The decellularized heart could support cardiomyocyte function as demonstrated by ESCs that exhibited beating three days after seeding17.\n\nDecellularized grafts can be modified to enhance their key functions. Immobilization of heparin to decellularized blood vessels using click chemistry reduced platelet adhesion and promoted EC attachment without altering the graft mechanical behavior18. Selective attachment of biological molecules is preferable to passive adsorption in attempting to compensate for damage to the extracellular matrix during removal of cells. Rather than harvest and remove cells from blood vessels, the extracellular matrix synthesized by cultured SMCs can be used to create decellularized vessels in a tubular polyglycolic acid scaffold19, thus providing a more controlled source of readily available extracellular matrix. A similar approach was used to create decellularized heart valves20, which were repopulated with cells eight weeks after implantation and performed better than decellularized valves.\n\nFor valve leaflets, the need to have regional variations in cell types and material properties was achieved using decellularized valves or using injection molding21 or three-dimensional bioprinting7 to fabricate specific three-dimensional shapes. Since decellularized tissue can be formed into hydrogels22 or electrospun, the use of different fabrication methods creates the possibility of precisely designing the tissue to be replaced.\n\nA novel approach to generate the entire TEBV in vivo involves taking advantage of the foreign body response and implanting a mandrel subcutaneously around which a tubular tissue grows over a four-week period23. Initially, the graft consisted of extracellular matrix and fibroblasts with a layer of M1 macrophages. After forming an end-to-end carotid anastomosis in the pig, the macrophages disappeared. After four weeks of grafting, the gene expression profile became similar to that of the carotid artery and fibroblasts adopted a contractile phenotype. The mechanical strength was very good but was less than values for actual vessels. This is a promising approach to develop engineered blood vessels, and other applications involve contracting SMCs (e.g. the bladder), which can be derived from fibroblasts involved in the foreign body response. Extending to other organ systems with specialized cells may prove difficult.\n\n\nStem cells for tissue engineering\n\niPSCs offer the potential to develop engineered tissues of individual human cardiovascular disease states and avoid ethical issues associated with ESCs. iPSCs can be induced to differentiate into a large number of cell types including cardiomyocytes, SMCs24,25, and ECs24. The formation of teratomas26 can be reduced using non-integrating methods27 and immunogenicity is low28. An exciting new development has been the creation of mouse iPSCs using small molecules that activate specific transcription factors29, although this approach has not yet been demonstrated with human iPSCs. A challenge with the use of iPSCs in tissue engineering is that differentiation is often limited and the resulting structures do not display a mature phenotype30,31.\n\nVascular cells can be obtained from iPSCs or ESCs by first activating the Wnt signaling pathway. Early activation of Wnt and β-catenin by inhibition of glycogen synthase kinase 3 (GSK3) before differentiation on surfaces with serum produces cardiomyocytes32. Following Wnt pathway activation with GSK3 inhibitors, ECs can be obtained by addition of vascular endothelial growth factor (VEGF) and forskolin, while SMCs can be obtained using platelet-derived growth factor-BB (PDGF-BB) and ActivinA25. Interestingly, a recent report indicated that by culturing murine iPSCs on gelatin-coated polycaprolactone nanofibrous scaffolds, Wnt/β-catenin can be transiently activated to induce differentiation towards cardiomyocytes33. Combining GSK3 inhibitors with specific modification of substrate properties may lead to more robust differentiation.\n\nOne week of electrical stimulation at 0.5, 1, or 2 Hz of human ESCs or iPSCs in three-dimensional engineered tissues facilitates differentiation to cardiomyocytes by producing hypertrophy, an increase in connexin-43 gap junctions, and increased expression of hERG, the potassium channel which regulates cardiomyocyte repolarization34. Some connexin-40 is expressed, indicating that rapidly conducting cells can be stimulated; however, it is not yet possible to regulate the relative expression of the various connexins though selection of a specific stimulation protocol. The stimulated cells responded to chronotropic drugs and the cells maintained synchrony to the rate of applied stimulation for two weeks after the stimulation ended.\n\nCardiomyocytes generated by selecting for Nkx2-5-positive cells among mouse iPSCs exhibit a number of markers found in mature cardiomyocytes, and the resting membrane potential approaches physiological levels35. Three-dimensional engineered tissues produced aligned cardiomyocytes that exhibited adherens and gap junctions, although the electrophysiological responses were similar to those exhibited by fetal cardiomyocytes35.\n\nHuman ESCs in three-dimensional patches showed extensive maturation and exhibited β-adrenergic responses in the physiological range6. Engineered cardiac tissue derived from human ESCs integrated into damaged mouse myocardium and formed a vasculature connected to the host blood supply after 28 days but did not improve heart function owing to extensive cell loss36. Alternatively, partial reprogramming of cardiac fibroblasts can be done using viral transfection of transcription factors, a cocktail of small molecules or microRNAs that activate key transcription factors (e.g. Mef2c, myocardin, and serum response factor). These approaches have yielded some success in producing spontaneously contracting cells, although the frequency of these cells among the population is low37.\n\nCulturing human cardiac myocytes derived from iPSCs on polydimethylsiloxane (PDMS) membranes coated with Matrigel for one week led to significant maturation of the cardiac cells in which the action potential upstroke velocity increased and conduction velocities were twice the value found when the cells were grown on Matrigel-coated glass coverslips38, although this value was still about 57% of the in vivo value. This increased maturation was due to a substantial increase in increased inward rectifier potassium and sodium inward current densities, elevated connexin-43 protein expression, hypertrophy of the cardiomyocytes, and increased cardiac troponin β1 integrin and focal adhesion kinase. The elastic modulus of the PDMS is approximately 4 MPa, much lower than the modulus of glass (~50 GPa), and the PDMS modulus is much greater than the modulus of cardiac tissue (0.1 MPa)39, suggesting that substrates with lower elastic modulus might enhance differentiation further. Modulating the substrate stiffness together with mechanical loading and electrical stimulation, which promote physiological force-frequency and force-length relations40, could produce cardiomyocytes with in vivo electrical and mechanical properties.\n\nThe use of small molecules to differentiate iPSCs has been used to create highly differentiated ECs that model the high transport resistance of brain ECs41. A number of cardiac disease models have been generated using iPSC technology and could replicate the response to cardiotoxic drugs using cells from various individuals42. The technology can also be used to assess the adaptive response to dilated cardiomyopathy. For example, cardiomyocytes derived from iPSCs of healthy individuals using small molecules exhibited many of the molecules involved in β-adrenergic signaling and isoproterenol treatment induced inotropic and chronotropic regulation of contractile function43. However, cardiomyocytes derived from iPSCs of individuals with dilated cardiomyopathy exhibited abnormal sarcomere structure and deficits in contractile force, calcium handling, and beat frequency after treatment with isoproterenol that was traced to overexpression of phosphodiesterases 2 and 3a43.\n\nTEBVs fabricated with SMCs derived from murine44 and human45 iPSCs maintained their differentiated phenotype after subcutaneous implantation for two weeks. Contractile TEBVs with SMCs differentiated from iPSCs developed from human foreskin fibroblasts and MSCs demonstrated intermediate and late SMC proteins46. While TEBVs derived from karyotypically normal human iPSC clones function well and express mid-differentiation markers SM-22α and calponin and secreted extracellular matrix, those derived from karyotypically abnormal clones exhibit senescence, shortened telomeres, and calcification4.\n\nHuman blood-derived ECs can be reprogrammed to SMCs by activating myocardin using a lentivirus system47. Functional TEBVs were produced with these cells that exhibited flow-mediated vasodilation and vasoconstriction in the presence of 1 µM phenylephrine47. While the vasoactivity was somewhat less than that of primary cells5, the results do show that TEBVs can be recreated with cells from a single donor.\n\n\nVascularization\n\nThe density and thickness of engineered tissues is limited by the transport of nutrients to the cells. Oxygen is often the limiting nutrient, since it is consumed at the highest rate and is critical for producing the energy needed for normal cell function. In vivo, capillary distances range from 15–50 µm depending on the cell density and the metabolic demands48. In vitro, cell densities are lower, but uniform cell densities can be achieved only for thicknesses of about 100 µm owing to consumption of oxygen in the engineered tissue. Perfusion can lead to somewhat thicker tissues. However, without its own microvascular network that could integrate with the host network after implantation, only thin tissue-engineered constructs can remain viable after implantation.\n\nWhile addition of VEGF can initiate the formation of new capillaries or branches from existing capillaries in vitro, the resulting structures are unstable and last at most a few days. New vessel formation involves several discrete stages. Initially, exogenous VEGF causes the release of matrix metalloproteinases, which degrade the extracellular matrix, enabling migration of the newly forming vessel buds. The newly forming vessel secretes growth factors to recruit mural cells, such as fibroblasts, pericytes, or SMCs, which interact with the newly formed microvessels, stabilizing them.\n\nAlthough MSCs can stabilize EC networks in vitro and exhibit pericyte-like behavior49, the heterogeneity of MSCs from various sources or by different isolation methods leads to variable responses50. The cells must be characterized and tested for their ability to stabilize networks when developing a system to create microvascular networks. iPSCs could provide a ready source of pericytes51, although SMCs or fibroblasts derived from iPSCs may be suitable.\n\nA potentially useful model system to identify conditions that promote vascularization of tissue-engineered systems involves creating microvascular networks in a synthetic extracellular matrix hydrogel. The hydrogel contains matrix metalloproteinase degradation sites and peptide sequences of extracellular matrix proteins to elicit specific cell binding52. Photopolymerization of polyethylene glycol gels enables straightforward incorporation of cells, and the networks are robust and sensitive to perfusion in the extracellular space52. The direction of flow is very critical for improving mass transfer and enabling microvessels to stabilize53. The extracellular matrix peptide sequences provided influence the extent of network formation, with addition of cell binding sequences from both fibronectin (RGD) and laminin (YIGSR) producing the most robust network formation in the hydrogel54. Adding macrophages enhanced new vessel formation in synthetic hydrogels, consistent with their role in vivo55. Other factors to enhance microvessel network formation in hydrogels involve regulating growth factor delivery56 and a hypoxic environment57.\n\nSeveral approaches have been taken to incorporate vascular networks into tissue-engineered constructs for implantation. When ECs were added with MSCs to decellularized heart tissue, vascular networks formed and enabled cell growth further into the construct than could be accomplished with MSCs alone17. The resulting network may have facilitated more effective fluid and nutrient transport throughout the decellularized tissue.\n\nEC cords show promise as a method to create functional microvascular networks in engineered constructs58,59. The cords are formed by mixing ECs and mural cells in collagen. After shrinkage by 50% in diameter over four hours, the cords are encased in fibrin and integrated into the tissue-engineered construct58. After implantation of cords into mice, capillaries formed within seven days and matured by 14 days. Red cells were observed in the lumen and an EC monolayer formed, defining the capillary border58. The capillaries involved both donor and host ECs. When the EC and MSC cords were added with hepatic construct, they improved key hepatocyte functions58. Cord diameters of 25, 75, and 250 µm all produced functional capillary networks, although smaller cords produced a higher density of vessels and the larger cords led to more dispersed vessels59. Mural cells were not necessary to form functioning capillaries after implantation, possibly owing to the involvement of host mural cells59. This approach can be used to control the density and geometry of the microvascular network, two properties that vary based on demand and function of the tissue.\n\n\nMicrophysiological systems\n\nHigh-throughput screens for function or to test drugs are being developed by integrating tissue engineering, microfluidics, and advanced methods of sensing. The National Center for Advancing Translational Science (NCATS) at NIH and the Defense Advanced Research Programs Association have led an effort to advance individual microphysiological systems and examine interactions among different organ systems. Microphysiological systems have been developed for the heart34,60, blood vessels5, microcirculation53, kidney61, gut62, lung63, liver64, skeletal muscle65, and female reproductive tract66. The small size of the systems reduces or eliminates mass transfer limitations, and function can be monitored with sensors or reporter systems. These systems have been developed using a combination of primary human cells and iPSCs. iPSCs provide the ability to create patient-specific cardiovascular disease models owing to their ability to maintain the disease phenotype post-differentiation67,68. Gene editing makes feasible isogenic controls for in vitro studies69.\n\nMicrophysiological systems based on the cardiovascular system reproduce key functions and known drug responses. Human endothelialized TEBVs with inner diameters of 500–800 µm exhibit a dose-dependent contraction in response to phenylephrine and a dose-dependent relaxation following exposure to acetylcholine over five weeks in culture5. The TEBVs elicited reversible activation to acute inflammatory stimulation by TNF-α, which was blocked by pre-treating the TEBVs with statins5 and consistent with the pleotropic effect that statins exert on ECs70.\n\nSeveral different microphysiological systems have been developed to model cardiac function. In one, cardiomyocytes are grown on poly(N-isopropylacrylamide) (PIPAAm) in a microfluidic chamber71. Forces exerted by contracting cardiomyocytes are determined from deformation of PIPAAm. As many as 28 PIPAAm cantilevers can be incorporated in one chip, and the fluidics enable easy exchange after drug or agonist exposure. This system was used to study Barth syndrome, an X-linked mutation of an acyltransferase essential for modification of cardiolipin. Individuals with this syndrome die within a year of birth due to heart failure and/or infection. This cardiac microphysiological system showed reduced contractile stresses by cardiomyocytes derived from Barth syndrome iPSCs72. Contractile stresses returned to normal for cells treated with a modified RNA that corrected the mutation and was improved after treatment with linoleic acid72, suggesting a novel treatment. An alternative approach to quantify contraction involves measuring strains using digital image correlation software to analyze the deformation of the engineered muscle34. To convert to stress, the stress-strain behavior of the muscle is needed.\n\nAnother system provided short transport distances and confined cardiomyocytes at high cell density in a microfluidic chamber60. The confinement barrier mimics the diffusive resistance of an endothelial monolayer but lacks the biochemical signals that arise from EC-cardiomyocyte interactions. Aligned and synchronously beating human cardiomyocytes derived from iPSCs were produced over a period of seven days. Cardiac cell motion was analyzed using custom software and was found to accurately represent cardiac cell responses to calcium channel and hERG blockers and β-adrenergic agonists and antagonists. The system can easily integrate the measurement of reporter fluorescence assays and analysis of the media after perfusion60.\n\n\nSummary and future directions\n\nNew technologies to promote cell differentiation and vascularize engineered constructs address key challenges in making viable engineered tissues that can be implanted. At the same time, decellularized tissues, either derived from organs and tissues or fabricated in the lab, make available an alternative approach to tissue engineering in which the implanted matrix serves as a substrate to guide cell repopulation and differentiation after implantation. Both approaches have aided our understanding of the complex interactions between cells and the extracellular matrix in producing a functional tissue.",
"appendix": "Competing interests\n\n\n\nThe author declares that he has no competing interests.\n\n\nGrant information\n\nThis work was supported, in part, by NIH grant UH3TR000505.\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\nI appreciate the many helpful conversations with my colleagues Nenad Bursac and William Kraus and students Cristina Fernandez and Leigh Atchison.\n\n\nReferences\n\nJones JR, Zhang SC: Engineering human cells and tissues through pluripotent stem cells. Curr Opin Biotechnol. 2016; 40: 133–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHarrison RH, St-Pierre JP, Stevens MM: Tissue engineering and regenerative medicine: a year in review. Tissue Eng Part B Rev. 2014; 20(1): 1–16. PubMed Abstract | Publisher Full Text\n\nWobma H, Vunjak-Novakovic G: Tissue Engineering and Regenerative Medicine 2015: A Year in Review. 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"id": "14065",
"date": "31 May 2016",
"name": "David Schaffer",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14066",
"date": "31 May 2016",
"name": "Gordana Vunjak-Novakovic",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14067",
"date": "31 May 2016",
"name": "Irving H Zucker",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1045
|
https://f1000research.com/articles/5-1044/v1
|
31 May 16
|
{
"type": "Review",
"title": "Recent advances in managing and understanding diabetic nephropathy",
"authors": [
"Sydney C.W. Tang",
"Gary C.W. Chan",
"Kar Neng Lai",
"Gary C.W. Chan",
"Kar Neng Lai"
],
"abstract": "Diabetic nephropathy is the commonest cause of end-stage renal disease in most developed economies. Current standard of care for diabetic nephropathy embraces stringent blood pressure control via blockade of the renin-angiotensin-aldosterone system and glycemia control. Recent understanding of the pathophysiology of diabetic nephropathy has led to the development of novel therapeutic options. This review article focuses on available data from landmark studies on the main therapeutic approaches and highlights some novel management strategies.",
"keywords": [
"Diabetic nephropathy",
"glycemia control"
],
"content": "Introduction\n\nDiabetic nephropathy (DN) affects approximately one-third of individuals with diabetes mellitus (DM) and carries with it considerable cardiovascular morbidity and mortality. Despite modern management of DM, the prevalence of this clinical entity continues to increase in association with an escalating diabetic population and, surprisingly, the excess mortality risk of DM is practically exclusively correlated with the occurrence of DN. Realistically, finding therapeutic modalities to stem this inexorable tide hinges upon a thorough understanding of the pathogenetic mechanisms leading to DN.\n\nRecent evidence shows that DN comprises a heavy inflammatory element triggered by metabolic disorders, protein overload, and hemodynamic abnormalities1–3. Although traditionally viewed to be glomerular in origin, emerging data suggest that the tubular epithelial cell plays an important role in orchestrating renal inflammation in DN. The activation of NF-κB and pro-inflammatory chemokines/cytokines in tubular epithelial cells were associated with the extent of the proteinuria and interstitial cell infiltration4. Targeting some of NF-κB-related inflammatory molecules may have therapeutic potential. For instance, blocking CCL2 has shown promise in preliminary clinical trials and will be discussed below. Another potentially important mediator of metabolic inflammation during DN is the Toll-like receptor (TLR). Overexpression of TLR2 and TLR4 in monocytes is positively correlated with hemoglobin A1c (HbA1c) levels in diabetic patients5, and TLR4 is also expressed in the renal tubules of human kidney biopsies of DN6. As blockade of TLR signaling has not yet been developed for clinical application, it will not be further discussed. Herein, we review the established therapeutic armamentarium and the progress in this emerging field, highlighting some novel management strategies arising from recent understanding of the mechanistic pathways leading to DN.\n\n\nCurrent standard of approach to diabetic nephropathy\n\nExtended observations from the EDIC (Epidemiology of Diabetes Interventions and Complications) study on the original Diabetes Control and Complications Trial cohort of type 1 diabetics clearly demonstrated a legacy effect of early intensive diabetic control beyond 18 years, with an overall risk reduction of 44% in developing chronic kidney disease (CKD) with estimated glomerular filtration rate (eGFR) lower than 60 ml/min/1.73m27–9. For type 2 diabetics, the UK Prospective Diabetes Study (UKPDS)10 with follow-up of 3,867 newly diagnosed patients showed that, compared with the conventional group (achieved HbA1c 7.9%), the risk in the intensive group (HbA1c 7.0%) was 12% lower for any diabetes-related endpoint; 10% lower for any diabetes-related death; and 6% lower for all-cause mortality. The majority of the lowered risk in any diabetes-related aggregate endpoint was attributable to a 25% risk reduction in microvascular endpoints. More recently, the ADVANCE (Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation) trial, that included 11,140 patients11, also demonstrated the value of tight glycemic control in terms of reduction of albuminuria (risk reduced by 9% and 30% for micro- and macro-albuminuria, respectively) and the risk of end-stage renal disease (ESRD, by 65%).\n\nThese encouraging data must be interpreted with caution, as reduction in albuminuria may be offset by the negative consequences of hypoglycemia from strict diabetic control. In the UKPDS10, patients in the intensive group had significantly more hypoglycemic episodes than those in the conventional group, regardless of whether data were analyzed by intent-to-treat or actual therapy. The ACCORD (Action to Control Cardiovascular Risk in Diabetes) trial was terminated early due to excess mortality in the intensive therapy arm (HbA1c target <6.0%) versus the standard arm (HbA1c 7.0–7.9%)12. Likewise, severe hypoglycemia observed in the ADVANCE cohort was linked to a range of adverse clinical effects, which prompted speculation on what constitutes optimal diabetic control13.\n\nThe American Association of Clinical Endocrinologists recommends an HbA1c target of <6.5%, while the American Diabetes Association sets a goal of HbA1c <7%, aiming to strike a balance between the risk of hypoglycemia and the clear benefit of renoprotection14.\n\nIn patients with DM, hypertension has long been known to be an independent, modifiable variable which predisposes individuals to the development and acceleration of micro- and macro-vascular problems. Prospective observational data from UK Prospective Diabetes Study 36 showed that, for every 10 mmHg reduction in systolic blood pressure, there was a decrease in all DM-related complications and death by 12% and 15%, respectively15. This is echoed by post-hoc analyses of 1,513 type 2 DM patients with confirmed DN and hypertension in the RENAAL (Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan) trial that demonstrated that the risk of ESRD or death was raised by 6.7% for each 10 mmHg increase in baseline systolic blood pressure16.\n\nBlockade of the renin-angiotensin system (RAS) using angiotensin-converting enzyme inhibitors (ACEi) or angiotensin II receptor blockers (ARB) is superior to using other anti-hypertensive agents in DN. They provide other renoprotective benefits beyond simply regulation of blood pressure, which are apparent from the results of the MARVAL (Micro-Albuminuria Reduction with Valsartan) study. For any given level of blood pressure reduction, after 24 weeks valsartan was shown to perform better than amlodipine in reducing micro-albuminuria (56% compared to 92% from baseline) in 332 type 2 DM individuals17. Treatment with ACEi was found to restrict development to macro-albuminuria by 60% in a meta-analysis of 698 non-hypertensive type 1 DM patients with micro-albuminuria. Additionally, an increased odds ratio of 3.07 (95% confidence interval [CI] 2.15 – 4.44; P < 0.001) for regression to normo-albuminuria was demonstrated18. Moreover, a sub-study of the IRMA-2 (Irbesartan in Patients with Type 2 Diabetes and Micro-albuminuria) trial showed that the reduction in micro-albuminuria by RAS blockade may persist, even after treatment withdrawal, which implies that glomerular structural normalization may be occurring19. In addition to the effects on micro-albuminuria, RAS blockade is equally effective in controlling macro-albuminuria20,21.\n\nAmeliorating albuminuria forms an integral treatment goal to reduce hard renal endpoints for RAS blockade. Irbesartan was found to decrease the risk of serum creatinine doubling and progression to ESRD by 33% and 23%, respectively, in the IDNT (Irbesartan Diabetic Nephropathy Trial) involving 1,715 hypertensive type 2 DN patients and a mean follow-up of 2.6 years22. Similar observations have arisen from the post-hoc analyses of RENAAL, in which a 50% decrease in albuminuria after 6 months of losartan treatment correlated with a 45% decreased risk for ESRD at 4 years of follow-up23. These findings recapitulate the renoprotective effect of captopril in type 1 diabetics with overt nephropathy20.\n\nThere is little direct comparison between ACEi and ARB and they appear to have comparable efficacy in DN, although intractable dry cough may be associated with ACE inhibition. These findings are reinforced by the DETAIL (Diabetics Exposed to Telmisartan and Enalapril) trial, a randomized clinical trial (RCT) comparing telmisartan to enalapril in 250 type 2 DN patients. After 5 years, the degree of glomerular filtration rate (GFR) decline, albuminuria and ESRD incidence were no different between the study arms24.\n\nIt must be borne in mind that secondary prevention trials have so far provided all existing data for RAS blockade. In addition, the use of the dihydropyridine class of calcium channel blockers (CCB) in the control group in some of the RCTs, such as MARVAL (17), could be a potential confounder, as this class of CCB is known to increase afferent arteriolar vasodilation and therefore may aggravate microalbuminuria in the control group. The National Kidney Foundation KDOQI clinical practice guidelines have not recommended using ACEi or ARB for the primary prevention of DN in normotensive individuals with normo-albuminuria25.\n\nThere is a theoretical pharmacologic basis for combining ACEi and ARB to maximize RAS blockade. In the CALM (Candesartan and Lisinopril Micro-albuminuria) study, a combination of candesartan and lisinopril was shown to lower micro-albuminuria more effectively than either drug alone at 12 weeks26. However, longer follow-up studies were never able to reproduce these short-term results. Moreover, no trial has as yet clearly demonstrated a more favorable renal outcome with dual RAS blockade. The findings from one RCT—ONTARGET (Ongoing Telmisartan Alone and in Combination with Ramipril Global Endpoint Trial), in which ramipril, telmisartan or both were administered to 25,620 high vascular risk patients (37.5% diabetics)—question the use of dual blockade, as combination therapy was shown to increase the composite outcome of dialysis, doubling of serum creatinine, and death (hazard ratio [HR] 1.09; 95% CI 1.01 – 1.18; P ≤ 0.037)27. The immediate response from the renal community was that ONTARGET was likely to be off target28. More recently, however, VA NEPHRON-D (Veterans Affairs Nephropathy in Diabetes) looked at 1,448 type 2 DN patients with eGFR 30–89.9 mL/min/1.73m2 treated with losartan alone or in combination with lisinopril29. After a median follow-up of just 2.2 years, the trial ended early due to no renal benefit being observed with dual therapy and an excessive risk of hyperkalemia (9.9% vs. 4.4%) and acute kidney injury (18% vs. 11%). In DN patients with more advanced CKD, dual RAS inhibition would carry an even greater risk. In general, therefore, combination therapy cannot be advised for DN management.\n\nApart from combining ACEi and ARB, aldosterone antagonism may be another approach to complementing RAS blockade. In fact, meta-analyses have demonstrated that a supplement of a mineralocorticoid receptor antagonist (MRA) given to those treated with ACEi or ARB produces a decrease in proteinuria in the CKD population30. Such beneficial effects were likewise observed in DN cohorts following administration of non-selective (spironolactone)31–33 and selective (eplerenone)34 MRA. However, several of the studies exploring the use of aldosterone antagonism in combination with RAS inhibition found evidence for a greater risk of hyperkalemia.\n\nFinerenone is a new nonsteroidal MRA with increased receptor selectivity compared to spironolactone and greater receptor affinity than eplerenone in vitro, along with a less frequent occurrence of hyperkalemia than spironolactone35. In a recent trial36 that recruited patients with type 2 DM and urine albumin-to-creatinine ratio (UACR) above 30 mg/g, finerenone added to ACEi or ARB produced a dose-dependent decrease in UACR without inducing hyperkalemia at day 90. The study had several important limitations37. For example, 60% of participants had GFR >60 ml/min/1.73m2, and consequently had a greatly decreased risk of hyperkalemia when compared with participants that had more severe renal disease. Additionally, two-thirds of the patients were receiving loop or thiazide diuretics, which facilitate kaliuresis. Finally, only a small drop in blood pressure was observed in those having the highest dose of finerenone, contrasting with earlier reports showing that steroidal MRAs lower blood pressure when combined with other medications, including RAS blockers. This might indicate a different mechanism of action of steroidal and nonsteroidal MRAs.\n\nStatins are the most widely used class of drug for lipid lowering in individuals with type 2 diabetes, reflecting the indisputable evidence that lowering of LDL cholesterol in individuals with type 2 diabetes is associated with reduced cardiovascular events and mortality. The role of lipid-lowering treatments in renoprotection for patients with diabetes, however, is debatable. In the Medical Research Council/British Heart Foundation (MRC/BHF) Heart Protection Study38, subgroup analysis for participants with diabetes, allocation to simvastatin (40 mg/day) significantly decreased the rise in serum creatinine values. Subjects with late stage CKD were not studied, as those with serum creatinine >200 umol/L were excluded from the trial. On the other hand, allocation to simvastatin plus ezetimibe in the Study of Heart and Renal Protection (SHARP)39 comprising 23% diabetic subjects did not produce significant reductions in any of the prespecified measures of renal disease progression among the subgroup of 6,247 nondialysis patients with a mean eGFR of 26.6 ml/min/1.73m2. Whether lipid lowering could only confer tangible renoprotection during early rather than late CKD requires further investigation.\n\nIn the Greek atorvastatin and coronary heart disease evaluation (GREACE)40 patients given atorvastatin had a significant reduction in urinary albumin excretion; however, separate analysis for type 2 diabetes was not included in the study. Such findings have been echoed recently by the PLANET I study41, in which treatment with atorvastatin 80 mg lowered UPCR substantially more than rosuvastatin 10 mg (-15·6%, 95% CI -28·3 to -0·5; p=0·043) or rosuvastatin 40 mg (-18·2%, -30·2 to -4·2; p=0·013). It must be cautioned that such doses of atorvastatin are unusually high for the average CKD patient.\n\n\nNovel therapeutic modalities\n\nDespite maximal RAS inhibition and other measures to control blood pressure and hyperglycemia, DN progression to ESRD remains intractable in many patients. Renewed understanding of the pathophysiology of DN has fueled the development of several potentially promising novel therapeutic options, and these are summarized below.\n\nCertain hypoglycemic agents have been shown to confer independent renoprotective effects beyond their hypoglycemic action. For instance, peroxisome proliferator activator receptor-gamma (PPAR-γ) agonists, also known as thiazolidinediones (TZD), have direct renoprotective effects in experimental models42. However, reports from clinical studies have been varied, with some achieving encouraging results by lowering proteinuria43–44, whilst some have demonstrated no meaningful effect45. Post-hoc analysis of the results of the PROactive (Prospective Pioglitazone Clinical Trial in Macro-vascular Events) study, which involved 5,238 DM subjects with macro-vascular complications, even reported a larger decrease in eGFR with pioglitazone46. Amongst the confusion, a meta-analysis of 15 TZD trials (10 with pioglitazone; 5 with rosiglitazone) which enrolled 2,860 patients did show a significant decline in albuminuria47. Apart from these surrogate end-points, however, there is still no data to support the fact that TZDs may improve hard renal outcomes, and several safety concerns have now been raised regarding these drugs, including heightened cardiovascular risks48,49 and malignancy50,51. With the current evidence, TZDs are unlikely to be a major player in the therapeutic armamentarium for DN.\n\nGlucagon-like peptide 1, an incretin which promotes insulin and suppresses glucagon release, is produced by the gut when food is ingested and it is degraded by dipeptidyl peptidase-4 (DPP-4)52. A novel group of hypoglycemic agents in the form of DPP-4 inhibitors have emerged in the treatment paradigm of DM, and experimental models have indicated possible renoprotective benefits53,54. Currently, data has only been obtained from a few clinical trials; however, in small, uncontrolled studies, 6 months of sitagliptin55 or 12 weeks of alogliptin56 lowered albuminuria in patients with type 2 DM. These findings must be interpreted with caution, as the sample size was small and treatment had prompted HbA1c to be lowered appropriately. Thus, it is difficult to delineate the role of the improved glycemic control in the reduction of albuminuria. However, the results of four phase III studies, comprising 217 patients with DN on RAS inhibition, indicated that 24 weeks of linagliptin significantly reduced albuminuria (32% reduction; 95% CI -42 to -21; P < 0.05), independent of HbA1c57. The encouraging findings regarding DPP-4 inhibitors, combined with their tolerability, weight neutral benefit and low risk of hypoglycemia58,59 have triggered further research into the gut-renal axis as a possible focus of future treatments60. Indeed, numerous clinical trials are currently underway to explore incretin-based therapies for retarding the progression of DN.\n\nVitamin D receptor (VDR) activators demonstrated anti-inflammatory and anti-proteinuric effects in animal models of DN61,62. Findings from the phase III VITAL (Selective Vitamin D Receptor Activation with Paricalcitol for Reduction of Albuminuria in Patients with Type 2 Diabetes) trial indicate that adjuvant paricalcitol at 2 μg/day lowers residual albuminuria in DN63. However, 42% of patients needed a reduced dose of paricalcitol due to poor tolerance, not to mention the additional drawback of the high cost of treatment. Therefore, concrete evidence demonstrating the successful use of VDR activators to retard the progression of DN is still awaited.\n\nApart from their ability to enhance urinary glucose excretion and aid glycemic control, SGLT-2 inhibitors appear to also promote an attractive cardiovascular portfolio that includes blood pressure and body weight optimization64–66. In the EMPA-REG study67 that has recruited over 7,000 type 2 diabetics at high cardiovascular risk, empagliflozin when added to standard care reduced the rates of death from cardiovascular causes (3.7%, vs. 5.9% in the placebo group; 38% relative risk reduction [RRR]), hospitalization for heart failure (2.7% and 4.1%, respectively; 35% RRR), and death from any cause (5.7% and 8.3%, respectively; 32% RRR). Unpublished data (presented at the American Society of Nephrology Kidney Week 2015 in San Diego) on renal outcomes are also promising, with significant reductions in new onset or worsening of nephropathy and the composite renal endpoints of doubling of serum creatinine, initiation of renal replacement therapy or death from renal cause.\n\nMonocyte chemoattractant protein-1 (MCP-1), also called C-C chemokine ligand 2 (CCL2), one of the ligands for C-C chemokine receptor type 2 (CCR2), has been implicated not only in insulin resistance but also in progressive renal injury, and has been suggested to be a potential marker of renal disease. In DN, MCP-1 overexpression plays an indispensable role in promoting monocyte and macrophage migration and activation68. CCX140-B is a small molecule CCR2 antagonist that inhibits CCR2 and blocks MCP-1-dependent monocyte activation and chemotaxis. Data from preclinical studies suggested that oral CCX140-B improved glycaemia and albuminuria in a mouse model of diabetes69.\n\nThe first evidence that CCR2 inhibition lowers albuminuria in DN came from a recent European study70. Patients with type 2 DM aged 18–75 years with UACR 100–3000 mg/g, eGFR ≥25 mL/min/1.73m2, and taking stable antidiabetic treatment and an ACEi or ARB for at least 8 weeks, were stratified to oral placebo, 5 mg CCX140-B, or 10 mg CCX140-B once a day. UACR changes from baseline during 52 weeks were -2% for placebo (95% CI -11% to 9%), -18% for 5 mg CCX140-B (-26% to -8%), and -11% for 10 mg CCX140-B (-20% to -1%). There was a -16% difference between 5 mg CCX140-B and placebo and a -10% difference between 10 mg CCX140-B and placebo, without significant difference in adverse events or renal events during the study. The data suggest that CCR2 inhibition with CCX140-B has albumin-lowering effects on top of current standard of care in patients with DN. Translation into hard evidence in follow-up studies that test whether CCX140-B also limits progression to end-stage renal disease is needed.\n\n\nConclusion\n\nDespite improved understanding of the pathophysiology of DN over the last 2 decades, an effective and specific treatment for this inexorable condition remains limited as the incidence of type 2 DM is predicted to continue an exponential upward trajectory, particularly in the developing world. The clinician is still equipped with no more than merely RAS blockers for control of blood pressure, various hypoglycemic agents for optimizing blood glucose and perhaps statins for controlling hyperlipidemia. Large-scale clinical trials that rode on the identification of emerging pathophysiologic pathways have met successes and tribulations [reviewed in reference 71] and we await the results of a number of further trials in the therapeutics of DN.",
"appendix": "Competing interests\n\n\n\nNone to declare.\n\n\nGrant information\n\nThis work was supported by grants from the National Natural Science Foundation of China (Grant no. 81570647), the National Basic Research Program of China 973 program no. 2012CB517600 (no. 2012CB517606), an Endowment Fund established for the “Yu Professorship in Nephrology” awarded to Sydney C.W. Tang, and generous donations from Mr. Winston Leung of Keen Union Investment Ltd, and Mr. Chan Kwok Keung of the Hong Kong Concrete and the Continental Cement and Gypsum Co. Ltd. We apologize to those investigators whose work was not cited here due to space limitation.\n\n\nReferences\n\nNavarro-González JF, Mora-Fernández C: The role of inflammatory cytokines in diabetic nephropathy. J Am Soc Nephrol. 2008; 19(3): 433–42. PubMed Abstract | Publisher Full Text\n\nWada J, Makino H: Inflammation and the pathogenesis of diabetic nephropathy. Clin Sci (Lond). 2013; 124(3): 139–52. 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N Engl J Med. 2013; 369(20): 1892–903. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nNavaneethan SD, Nigwekar SU, Sehgal AR, et al.: Aldosterone antagonists for preventing the progression of chronic kidney disease: a systematic review and meta-analysis. Clin J Am Soc Nephrol. 2009; 4(3): 542–51. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchjoedt KJ, Rossing K, Juhl TR, et al.: Beneficial impact of spironolactone on nephrotic range albuminuria in diabetic nephropathy. Kidney Int. 2006; 70(3): 536–42. PubMed Abstract | Publisher Full Text\n\nMehdi UF, Adams-Huet B, Raskin P, et al.: Addition of angiotensin receptor blockade or mineralocorticoid antagonism to maximal angiotensin-converting enzyme inhibition in diabetic nephropathy. J Am Soc Nephrol. 2009; 20(12): 2641–50. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRachmani R, Slavachevsky I, Amit M, et al.: The effect of spironolactone, cilazapril and their combination on albuminuria in patients with hypertension and diabetic nephropathy is independent of blood pressure reduction: a randomized controlled study. Diabet Med. 2004; 21(5): 471–5. PubMed Abstract | Publisher Full Text\n\nEpstein M, Williams GH, Weinberger M, et al.: Selective aldosterone blockade with eplerenone reduces albuminuria in patients with type 2 diabetes. Clin J Am Soc Nephrol. 2006; 1(5): 940–51. PubMed Abstract | Publisher Full Text\n\nPitt B, Kober L, Ponikowski P, et al.: Safety and tolerability of the novel non-steroidal mineralocorticoid receptor antagonist BAY 94-8862 in patients with chronic heart failure and mild or moderate chronic kidney disease: a randomized, double-blind trial. Eur Heart J. 2013; 34(31): 2453–63. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBakris GL, Agarwal R, Chan JC, et al.: Effect of Finerenone on Albuminuria in Patients With Diabetic Nephropathy: A Randomized Clinical Trial. JAMA. 2015; 314(9): 884–94. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWeir MR: Diabetic nephropathy: Nonsteroidal MRA added to RAS blockade reduces albuminuria. Nat Rev Nephrol. 2015; 11(12): 691–2. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCollins R, Armitage J, Parish S, et al.: MRC/BHF Heart Protection Study of cholesterol-lowering with simvastatin in 5963 people with diabetes: a randomised placebo-controlled trial. Lancet. 2003; 361(9374): 2005–16. PubMed Abstract | Publisher Full Text\n\nBaigent C, Landray MJ, Reith C, et al.: The effects of lowering LDL cholesterol with simvastatin plus ezetimibe in patients with chronic kidney disease (Study of Heart and Renal Protection): a randomised placebo-controlled trial. Lancet. 2011; 377(9784): 2181–92. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nAthyros VG, Mikhailidis DP, Papageorgiou AA, et al.: The effect of statins versus untreated dyslipidaemia on renal function in patients with coronary heart disease. A subgroup analysis of the Greek atorvastatin and coronary heart disease evaluation (GREACE) study. J Clin Pathol. 2004; 57(7): 728–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Zeeuw D, Anzalone DA, Cain VA, et al.: Renal effects of atorvastatin and rosuvastatin in patients with diabetes who have progressive renal disease (PLANET I): a randomised clinical trial. Lancet Diabetes Endocrinol. 2015; 3(3): 181–90. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nIsshiki K, Haneda M, Koya D, et al.: Thiazolidinedione compounds ameliorate glomerular dysfunction independent of their insulin-sensitizing action in diabetic rats. Diabetes. 2000; 49(6): 1022–32. PubMed Abstract | Publisher Full Text\n\nBakris GL, Ruilope LM, McMorn SO, et al.: Rosiglitazone reduces microalbuminuria and blood pressure independently of glycemia in type 2 diabetes patients with microalbuminuria. J Hypertens. 2006; 24(10): 2047–55. PubMed Abstract | Publisher Full Text\n\nHanefeld M, Brunetti P, Schernthaner GH, et al.: One-year glycemic control with a sulfonylurea plus pioglitazone versus a sulfonylurea plus metformin in patients with type 2 diabetes. Diabetes Care. 2004; 27(1): 141–7. PubMed Abstract | Publisher Full Text\n\nAgarwal R, Saha C, Battiwala M, et al.: A pilot randomized controlled trial of renal protection with pioglitazone in diabetic nephropathy. Kidney Int. 2005; 68(1): 285–92. PubMed Abstract | Publisher Full Text\n\nSchneider CA, Ferrannini E, Defronzo R, et al.: Effect of pioglitazone on cardiovascular outcome in diabetes and chronic kidney disease. J Am Soc Nephrol. 2008; 19(1): 182–7. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSarafidis PA, Stafylas PC, Georgianos PI, et al.: Effect of thiazolidinediones on albuminuria and proteinuria in diabetes: a meta-analysis. Am J Kidney Dis. 2010; 55(5): 835–47. PubMed Abstract | Publisher Full Text\n\nNissen SE, Wolski K: Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes. N Engl J Med. 2007; 356(24): 2457–71. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGraham DJ, Ouellet-Hellstrom R, MaCurdy TE, et al.: Risk of acute myocardial infarction, stroke, heart failure, and death in elderly Medicare patients treated with rosiglitazone or pioglitazone. JAMA. 2010; 304(4): 411–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTurner RM, Kwok CS, Chen-Turner C, et al.: Thiazolidinediones and associated risk of bladder cancer: a systematic review and meta-analysis. Br J Clin Pharmacol. 2014; 78(2): 258–73. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nColmers IN, Bowker SL, Majumdar SR, et al.: Use of thiazolidinediones and the risk of bladder cancer among people with type 2 diabetes: a meta-analysis. CMAJ. 2012; 184(12): E675–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartin JH, Deacon CF, Gorrell MD, et al.: Incretin-based therapies--review of the physiology, pharmacology and emerging clinical experience. Intern Med J. 2011; 41(4): 299–307. PubMed Abstract | Publisher Full Text\n\nLiu WJ, Xie SH, Liu YN, et al.: Dipeptidyl peptidase IV inhibitor attenuates kidney injury in streptozotocin-induced diabetic rats. J Pharmacol Exp Ther. 2012; 340(2): 248–55. PubMed Abstract | Publisher Full Text\n\nAlter ML, Ott IM, von Websky K, et al.: DPP-4 inhibition on top of angiotensin receptor blockade offers a new therapeutic approach for diabetic nephropathy. Kidney Blood Press Res. 2012; 36(1): 119–30. PubMed Abstract | Publisher Full Text\n\nHattori S: Sitagliptin reduces albuminuria in patients with type 2 diabetes. Endocr J. 2011; 58(1): 69–73. PubMed Abstract | Publisher Full Text\n\nSakata K, Hayakawa M, Yano Y, et al.: Efficacy of alogliptin, a dipeptidyl peptidase-4 inhibitor, on glucose parameters, the activity of the advanced glycation end product (AGE) - receptor for AGE (RAGE) axis and albuminuria in Japanese type 2 diabetes. Diabetes Metab Res Rev. 2013; 29(8): 624–30. PubMed Abstract | Publisher Full Text\n\nGroop PH, Cooper ME, Perkovic V, et al.: Linagliptin lowers albuminuria on top of recommended standard treatment in patients with type 2 diabetes and renal dysfunction. Diabetes Care. 2013; 36(11): 3460–8. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSchernthaner G, Barnett AH, Emser A, et al.: Safety and tolerability of linagliptin: a pooled analysis of data from randomized controlled trials in 3572 patients with type 2 diabetes mellitus. Diabetes Obes Metab. 2012; 14(5): 470–8. PubMed Abstract | Publisher Full Text\n\nLehrke M, Marx N, Patel S, et al.: Safety and Tolerability of Linagliptin in Patients With Type 2 Diabetes: A Comprehensive Pooled Analysis of 22 Placebo-controlled Studies. Clin Ther. 2014; 36(8): 1130–46. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMuskiet MH, Smits MM, Morsink LM, et al.: The gut-renal axis: do incretin-based agents confer renoprotection in diabetes? Nat Rev Nephrol. 2014; 10(2): 88–103. PubMed Abstract | Publisher Full Text\n\nSanchez-Niño M, Bozic MD, Córdoba-Lanús E, et al.: Beyond proteinuria: VDR activation reduces renal inflammation in experimental diabetic nephropathy. Am J Physiol Renal Physiol. 2012; 302(6): F647–57. PubMed Abstract | Publisher Full Text\n\nZhang Z, Zhang Y, Ning G, et al.: Combination therapy with AT1 blocker and vitamin D analog markedly ameliorates diabetic nephropathy: blockade of compensatory renin increase. Proc Natl Acad Sci U S A. 2008; 105(41): 15896–901. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nde Zeeuw D, Agarwal R, Amdahl M, et al.: Selective vitamin D receptor activation with paricalcitol for reduction of albuminuria in patients with type 2 diabetes (VITAL study): a randomised controlled trial. Lancet. 2010; 376(9752): 1543–51. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBarnett AH, Mithal A, Manassie J, et al.: Efficacy and safety of empagliflozin added to existing antidiabetes treatment in patients with type 2 diabetes and chronic kidney disease: a randomised, double-blind, placebo-controlled trial. Lancet Diabetes Endocrinol. 2014; 2(5): 369–84. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTahrani AA, Barnett AH, Bailey CJ: SGLT inhibitors in management of diabetes. Lancet Diabetes Endocrinol. 2013; 1(2): 140–51. PubMed Abstract | Publisher Full Text\n\nMusso G, Gambino R, Cassader M, et al.: A novel approach to control hyperglycemia in type 2 diabetes: sodium glucose co-transport (SGLT) inhibitors: systematic review and meta-analysis of randomized trials. Ann Med. 2012; 44(4): 375–93. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nZinman B, Wanner C, Lachin JM, et al.: Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes. N Engl J Med. 2015; 373(22): 2117–28. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTang SC, Lai KN: The pathogenic role of the renal proximal tubular cell in diabetic nephropathy. Nephrol Dial Transplant. 2012; 27(8): 3049–56. PubMed Abstract | Publisher Full Text\n\nSullivan T, Miao Z, Dairaghi DJ, et al.: CCR2 antagonist CCX140-B provides renal and glycemic benefits in diabetic transgenic human CCR2 knockin mice. Am J Physiol Renal Physiol. 2013; 305(9): F1288–97. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nde Zeeuw D, Bekker P, Henkel E, et al.: The effect of CCR2 inhibitor CCX140-B on residual albuminuria in patients with type 2 diabetes and nephropathy: a randomised trial. Lancet Diabetes Endocrinol. 2015; 3(9): 687–96. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nChan GC, Tang SC: Diabetic nephropathy: landmark clinical trials and tribulations. Nephrol Dial Transplant. 2016; 31(3): 359–68. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "14060",
"date": "31 May 2016",
"name": "Bruce M. Hendry",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14061",
"date": "31 May 2016",
"name": "York PC Pei",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14062",
"date": "31 May 2016",
"name": "Todd S 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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1044
|
https://f1000research.com/articles/5-1019/v1
|
27 May 16
|
{
"type": "Review",
"title": "Diabetic macular edema: it is more than just VEGF",
"authors": [
"Michael A. Singer",
"Daniel S. Kermany",
"Jana Waters",
"Michael E. Jansen",
"Lyndon Tyler",
"Daniel S. Kermany",
"Jana Waters",
"Michael E. Jansen",
"Lyndon Tyler"
],
"abstract": "Diabetic macular edema is a serious visual complication of diabetic retinopathy. This article reviews the history of previous and current therapies, including laser therapy, anti-vascular endothelial growth factor agents, and corticosteroids, that have been used to treat this condition. In addition, it proposes new ways to use them in combination in order to decrease treatment burden and potentially address other causes besides vascular endothelial growth factor for diabetic macular edema.",
"keywords": [
"macular edema",
"VEGF",
"Diabetic macular edema",
"Laser photocoagulation"
],
"content": "Introduction\n\nDiabetic macular edema (DME) is a common complication of diabetes and the leading cause of vision loss in adults1. Macular edema occurs when high blood glucose damages the small capillaries supplying blood to the retina. This breakdown of blood vessels leads to the extravasation of blood and its solutes from the capillaries to the extracellular space under the macula, the central area of the retina, and causes it to thicken and swell (edema). The macula holds tightly packed cones that provide sharp central vision, enabling the person to see vivid detail and color. In this review, we briefly discuss the treatment options currently available for the treatment of macular edema and we review the rationale for emerging agents, many of which are currently being evaluated in clinical trials.\n\n\nTreatments\n\nLaser photocoagulation has been the gold standard for the treatment of DME for the past two decades. Laser treatment involves placing tiny laser burns within thickened areas of the retina in both direct (focal) treatment of microaneurysms and scattered spots in other areas of edema (grid). Laser photocoagulation involves the application of a precise and directed high-energy laser to the retina, and the heat generated as it is absorbed into the tissue causes clotting of the blood vessels and leads to the localized destruction of the tissue. These burns in the light-sensitive membrane in the back of the eye serve to destroy the diseased areas of the tissue and to seal off the damaging blood vessels that threaten vision.\n\nThis treatment for macular edema is very prevalent since it is a quick and cost-effective procedure that is usually completed after one session without the risk of endophthalmitis that an intravitreal injection poses. However, laser photocoagulation focuses on the symptoms of edema instead of addressing the retinopathy. The benefits to visual acuity are unremarkable, as there is only a 50% reduction in vision loss and vision already lost cannot be regained using laser photocoagulation. Lesions on the retina left by the destructive laser have been observed to expand over time. At 2 years, laser sears increased 50% per year and 4.6% a year afterwards, and 11 out of 203 patients experienced foveal encroachment. Still, laser photocoagulation is a highly effective treatment for macular edema and is still a feasible option for those unresponsive to anti-vascular endothelial growth factor (anti-VEGF) treatments2.\n\nThe VEGF family is the most critical with regard to the pathogenesis of diabetic retinopathy owing to its signaling the induction of angiogenesis as well as increasing vascular permeability. Because of VEGF’s central role in the pathogenesis of DME, VEGF antagonists are a logical choice for therapy. The first anti-VEGF agent used for ophthalmology was pegaptanib (OSI Pharmaceuticals, Long Island, NY, USA) but was replaced by the development of ranibizumab (Genentech, Inc., South San Francisco, CA, USA). In the randomized clinical trial Protocol I, researchers observed that ranibizumab triples the mean change in visual acuity compared to the corticosteroid triamcinolone, and in 1 year the same steroid showed a visual acuity loss three times higher than that produced by the VEGF antagonist3. In addition, the RISE and RIDE studies demonstrated that 39.2% of patients had 15-letter gains in visual acuity and a mean improvement of 12.4 letters vs. sham over 24 monthly injections and were the pivotal studies that allowed approval of ranibizumab by the US Food and Drug Administration4.\n\nBevacizumab (Genentech, Inc., South San Francisco, CA, USA) is another VEGF antagonist that has come into widespread clinical use in the treatment of retinal disease. The BOLT study compared bevacizumab vs. macular laser in patients with DME. The bevacizumab arm gained a median of 9 ETDRS letters vs. 2.5 letters of laser treatment (P=0.005), with a mean gain of 8.6 letters for bevacizumab vs. a mean loss of 0.5 letters for laser. Forty-nine percent of patients gained 10 or more letters (P=0.001) and 32% gained at least 15 letters (P=0.004) for bevacizumab vs. 7% and 4% for MLT. The percentage who lost fewer than 15 letters in the laser arm was 86% vs. 100% for bevacizumab (P=0.03)5.\n\nAflibercept is the most recent anti-VEGF medication approved to treat DME. In the randomized clinical studies VIVID and VISTA, researchers compared intravitreal aflibercept injections, which have recently demonstrated clinically equivalent efficacy to monthly ranibizumab in neovascular age-related macular degeneration, to laser monotherapy for the treatment of DME. The results of the trials demonstrated that aflibercept, given either every 4 weeks or every 8 weeks (after five initial monthly doses), is superior to laser and results in 10.7–12.4 letters gained at 1 year. In addition, 32–41% of patients gained 15 letters at 1 year as well6. These visual acuity results indicate that a large portion of patients with DME may be effectively treated with dosing every 8 weeks compared to the monthly injections of other anti-VEGF agents.\n\nHowever, it is widely accepted that the systemic use of anti-VEGF agents results in an increased risk for arterial thromboembolic events, obstructions of a blood vessel caused by a blood clot that has become dislodged from another point in the circulation, which can show a higher incidence of stroke among patients receiving this therapy. However, it is noteworthy that no significant increased rate of death, stroke, or myocardial infarction was seen in the RISE and RIDE or VIVID and VISTA groups.\n\nAbout 50% of patients with DME experience only a moderate reduction of edema and improvement in vision from VEGF antagonists alone. However, for most clinicians, it takes many months or years to determine the need for a switch in therapy. The EARLY analysis was a post hoc analysis of Protocol I of the diabetic retinopathy clinical research group7,8. This analysis looked at the two arms of patients who received ranibizumab and looked at vision at 12 months and 3 years. The study showed that patients could be divided into three groups: those who were good responders with 10 or more letters of improvement, those who were fair responders with 5 to 9 letters of improvement, and those who were suboptimal responders with 5 or fewer letters of improvement. The study showed that the best-corrected visual acuity (BCVA) response after three anti-VEGF injections (12 weeks) is a strong predictor of long-term BCVA response at 12 months and 3 years. This study demonstrated that physicians can recognize suboptimal DME responders much earlier in the treatment cycle and should consider different therapies in patients who are suboptimal responders.\n\nMounting evidence exists to show that inflammation is a significant aspect of the pathogenesis of DME. Leukocytes in the blood release a variety of cytokines and chemokines that significantly increase vascular permeability, leading to more fluid build-up under the retina. These cytokines also carry VEGFs, which can aggravate and worsen macular edema by promoting angiogenesis9.\n\nCorticosteroids have shown the ability to lower inflammatory mediators and VEGF, while anti-VEGF therapy treats only the VEGF portion. Anti-VEGF treatment does not work for all patients; 50% of patients respond significantly and quickly, 25% of patients have an intermediate response, and 25% of patients do not respond to anti-VEGF treatment10. Steroids have been shown to lower the central subfield thickness (CST) and improve visual acuity for suboptimal responders to anti-VEGF and pseudophakics. Corticosteroids also appear to be effective for both chronic and treatment-naive macular edema, while anti-VEGF therapy is seen to be a less effective treatment for chronic DME.\n\nThere are currently two approved corticosteroid therapies for DME: dexamethasone (Ozurdex, Allergan) and fluocinolone (Iluvein, Alimera). The MEAD study showed that dexamethasone intravitreal implant was able to improve vision in patients over a 3-year period. Patient vision improved overall by 4 letters and pseudophakic patients improved by 6 letters over a year period. In the MEAD study, there was a nearly 60% frequency of cataract surgery, and 40% of patients on steroids were later prescribed medication for intraocular pressure (IOP). However, patients’ increase in IOP usually peaked at 6–8 weeks and then returned to baseline by the end of 4 months, and one patient required IOP-lowering surgery11.\n\nThe advantage of corticosteroid delayed delivery systems, such as dexamethasone intravitreal implants, is that patients require fewer treatments compared to the monthly injections required by most anti-VEGF agents. The newest steroid to be approved is the fluocinolone implant. The FAME study showed that the fluocinolone implant caused an improvement of ≥15 letters in 28.7% of patients in the study group vs. 18.9% in sham eyes and a 6-letter improvement in vision at 24 months. IOP medications were required in 42% of patients with seven patients requiring IOP-lowering surgery12.\n\nAlthough anti-VEGF medication remains the mainstay of therapy for DME, there are many cases for which anti-VEGF therapy alone is not adequate enough to control the macular edema. This should not be unexpected, as the clinical trials were able to significantly improve vision in fewer than 50% of patients4–6,13. VEGF is a logical drug target that treats DME well, but it requires monthly retreatment to maintain efficacy and it does not address additional inflammatory cytokines upregulated in DME. It is in these patients that supplementing with therapies that work using a different mode of action may be of value. Diabetic maculopathy is a combination of both VEGF-mediated factors as well as inflammatory mediators. Corticosteroids decrease inflammatory cytokines and have a modest anti-VEGF effect, while anti-VEGF agents have a modest anti-inflammatory effect9. Using a corticosteroid in combination with an anti-VEGF agent allows the patient to benefit with increased efficacy as well as increased duration of effect. As the category of sustained release steroids increases, the physician’s arsenal of managing these “hard to treat” patients increases as well.\n\nA 12-month randomized study of eyes with persistent DME assessed the efficacy of a corticosteroid (dexamethasone) delivery system as an adjunct to the VEGF antagonist bevacizumab compared with continued bevacizumab monotherapy14. After 12 months, it was observed that while ultimately there was no difference in vision, there were differences in vision at different monthly time points and the optical coherence tomography (OCT) CST was significantly better in the combination group, with a “sawtooth” effect14. Subgroup analysis suggested that the greatest benefit of dexamethasone implant was in the group with the most bevacizumab injections prior to enrollment in the study14. In conclusion, although visual acuity changes are not superior to continued bevacizumab monotherapy, dexamethasone combined with bevacizumab significantly improves visual acuity and significantly improves macular morphology in eyes with refractive chronic DME.\n\nIn another study, researchers explored the effect and safety of fluocinolone acetonide in chronic DME patients considered insufficiently responsive to available therapies with or without intravitreal corticosteroid therapy15. The study covered 12 patients who received a single injection of fluocinolone acetonide and were followed for 6 months. Out of the 11 patients who completed the study, nine maintained or improved their best-corrected visual acuity from baseline, and the 11 patients experienced an average decrease in CST of 300.6 microns from baseline. However, there are no good data on combination therapy with the implant, but it may be appropriate for patients with suboptimal edema reduction with anti-VEGF monotherapy15.\n\nCombination therapy is a rational approach to battling DME, as it targets VEGF-mediated angiogenesis while combating the multiple factors in the inflammatory cascade. Combination therapy may be an alternative for patients who are unresponsive or have a suboptimal response to VEGF antagonists by providing a sustained duration of action, which translates into increased efficacy and convenience. However, there are potential adverse effects, such as cataracts and elevated IOP, that need to be considered.\n\n\nAbbreviations\n\nBCVA, best-corrected visual acuity; CST, central subfield thickness; DME, diabetic macular edema; IOP, intraocular pressure; OCT, optical coherence tomography; VEGF, vascular endothelial growth factor.",
"appendix": "Competing interests\n\n\n\nMichael A. Singer has served as a researcher for Allergan, Genentech, Regeneron, Ampio, Aerpio, Santen, Neurotech, and Optos; a consultant for Allergan, Genentech, Ampio, and Aerpio; and a speaker for Allergan, Genentech, Regeneron, and Ampio.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMoss SE, Klein R, Klein BE: The 14-year incidence of visual loss in a diabetic population. Ophthalmology. 1998; 105(6): 998–1003. PubMed Abstract | Publisher Full Text\n\nPhotocoagulation for diabetic macular edema. Early Treatment Diabetic Retinopathy Study report number 1. Early Treatment Diabetic Retinopathy Study research group. Arch Ophthalmol. 1985; 103(12): 1796–806. PubMed Abstract | Publisher Full Text\n\nDiabetic Retinopathy Clinical Research Network, Elman MJ, Aiello LP, et al.: Randomized trial evaluating ranibizumab plus prompt or deferred laser or triamcinolone plus prompt laser for diabetic macular edema. Ophthalmology. 2010; 117(6): 1064–1077.e35. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nNguyen QD, Brown DM, Marcus DM, et al.: Ranibizumab for diabetic macular edema: results from 2 phase III randomized trials: RISE and RIDE. Ophthalmology. 2012; 119(4): 789–801. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRajendram R, Fraser-Bell S, Kaines A, et al.: A 2-year prospective randomized controlled trial of intravitreal bevacizumab or laser therapy (BOLT) in the management of diabetic macular edema: 24-month data: report 3. Arch Ophthalmol. 2012; 130(8): 972–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBrown DM, Schmidt-Erfurth U, Do DV, et al.: Intravitreal Aflibercept for Diabetic Macular Edema: 100-Week Results From the VISTA and VIVID Studies. Ophthalmology. 2015; 122(10): 2044–52. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDugel P: EARLY: Anti-VEGF treatment response at week 12 and long term outcomes in DME. Paper presented at the American Academy of Ophthalmology Retina Subspecialty Day; November 13–17, 2015; Las Vegas, NV.\n\nGonzales V, Augustin A, Campbell J, et al.: EARLY: Anti-VEGF treatment response at week 12 and long term outcomes in DME. Paper presented at World Ophthalmology Congress; February 5–9, 2016; Guadalajara, Mexico.\n\nRangasamy S, McGuire PG, Das A: Diabetic retinopathy and inflammation: novel therapeutic targets. Middle East Afr J Ophthalmol. 2012; 19(1): 52–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBressler SB, Qin H, Beck RW, et al.: Factors associated with changes in visual acuity and central subfield thickness at 1 year after treatment for diabetic macular edema with ranibizumab. Arch Ophthalmol. 2012; 130(9): 1153–61. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBoyer DS, Yoon YH, Belfort R Jr, et al.: Three-year, randomized, sham-controlled trial of dexamethasone intravitreal implant in patients with diabetic macular edema. Ophthalmology. 2014; 121(10): 1904–14. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCampochiaro PA, Brown DM, Pearson A, et al.: Sustained delivery fluocinolone acetonide vitreous inserts provide benefit for at least 3 years in patients with diabetic macular edema. Ophthalmology. 2012; 119(10): 2125–32. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDiabetic Retinopathy Clinical Research Network: A randomized trial comparing intravitreal triamcinolone acetonide and focal/grid photocoagulation for diabetic macular edema. Ophthalmology. 2008; 115(9): 1447–9, 1449.e1–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaturi RK, Bleau L, Saunders J, et al.: A 12-month, single-masked, randomized controlled study of eyes with persistent diabetic macular edema after multiple anti-VEGF injections to assess the efficacy of the dexamethasone-delayed delivery system as an adjunct to bevacizumab compared with continued bevacizumab monotherapy. Retina. 2015; 35(8): 1604–14. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nFigueira J, et al.: RESPOND study: Interim six month results: Presented at the 2015 Euroretina Conference, Nice, France."
}
|
[
{
"id": "14058",
"date": "27 May 2016",
"name": "Robert Bhisitkul",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14059",
"date": "27 May 2016",
"name": "Szilárd Kiss",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1019
|
https://f1000research.com/articles/5-1018/v1
|
27 May 16
|
{
"type": "Review",
"title": "Improving outcome of sensorimotor functions after traumatic spinal cord injury",
"authors": [
"Volker Dietz"
],
"abstract": "In the rehabilitation of a patient suffering a spinal cord injury (SCI), the exploitation of neuroplasticity is well established. It can be facilitated through the training of functional movements with technical assistance as needed and can improve outcome after an SCI. The success of such training in individuals with incomplete SCI critically depends on the presence of physiological proprioceptive input to the spinal cord leading to meaningful muscle activations during movement performances. Some actual preclinical approaches to restore function by compensating for the loss of descending input to spinal networks following complete/incomplete SCI are critically discussed in this report. Electrical and pharmacological stimulation of spinal neural networks is still in the experimental stage, and despite promising repair studies in animal models, translations to humans up to now have not been convincing. It is possible that a combination of techniques targeting the promotion of axonal regeneration is necessary to advance the restoration of function. In the future, refinement of animal models according to clinical conditions and requirements may contribute to greater translational success.",
"keywords": [
"Spinal cord injury",
"trauma",
"treatment",
"spinal cord repair",
"neuroprotective",
"neuroregenerative"
],
"content": "Introduction\n\nOver the last few years, several approaches to improving the outcome of sensorimotor functions after spinal cord injury (SCI) have been established. These concern approaches to treating an acute traumatic SCI, exploiting neuroplasticity, and repairing damaged spinal cord by neuroprotective and neuroregenerative substances. For the latter approaches, a great number of animal studies have shown promising potential to translate the repair of the damaged spinal cord tracts to human SCI. In contrast, little progress has been made to treat impairment of the autonomic system, which impedes quality of life more than the ability of walking1. Nevertheless, this short opinion paper is limited in its scope, i.e. it focuses on a few actual approaches to restoring sensorimotor functions after an SCI and intends to point out some of the problems in translating preclinical animal work to the clinic. In addition, it is thought to be selective and complementary to another review, ‘Recent advances in managing a spinal cord injury secondary to trauma’, provided by Fehlings’ group2. The latter review discusses in more detail new approaches that are intended to be introduced into the clinic. Furthermore, a broader review about actual approaches to improving the outcome of sensorimotor functions after an SCI was recently published elsewhere3.\n\n\nManagement of acute injury\n\nThe actual treatment for SCI is focused on early decompression of the injured spinal cord, leading to an improved outcome4. This approach is convincing and well established. The limitations are twofold: life-threatening complications can have a higher priority, and after an accident patients are frequently not directly transferred to a hospital that can provide adequate spine surgery. The consequences are inevitable delays. Nevertheless, there are clear indications to perform decompression surgery, e.g. by expansion duroplasty to improve circulation in the damaged spinal cord5. Furthermore, by the application of new techniques of neuromonitoring from the injury site to guide the management of blood pressure, for example, and/or the preservation of spinal cord perfusion pressure at the injury site6, additional damage might be avoided.\n\n\nRehabilitation: exploitation of neuroplasticity\n\nGreat advances in the last few decades to exploit neuroplasticity, as well as the associated development of neurorehabilitation technology, as an important part of neurorehabilitation have become well established3. Actual training approaches are directed to the activation of specific receptors necessary to lead to physiological limb activation7 required for an improved outcome of function. Functional training programs based on animal experiments8 dominate this part of rehabilitation. Furthermore, a spontaneously occurring neural re-organisation takes place for the compensation of paresis, such as the development of spastic muscle tone9. This can be facilitated and adjusted by functional training.\n\nDuring the last 20 years, functional training programs have led to the rapid development of neurorehabilitation technology (for review, see 10). Today, the training of functional movements of upper and lower limbs becomes assisted by an increasing number of robotic devices in combination with virtual reality programs, which facilitate training, provide feedback information, and allow longer training times (for review, see 11).\n\n\nSpinal cord repair: neuroprotection and neuroregeneration\n\nThe ultimate goal in treating SCI and improving function would in any case be spinal cord repair. A large number of neuroprotective and neuroregenerative agents exist and frequently show positive effects in animal models of SCI. However, the actual proven effects of these approaches are rather disappointing. Pilot studies and Cochrane reviews12 indicate rather small beneficial effects of such agents (e.g. methylprednisolone13). For example, an increase of four motor score points means functionally almost nothing, especially if considered with respect to possible side effects of such treatment (e.g. respiration problems and infections). Today, none of these substances are applied routinely in SCI patients (at least in Europe); early administration of methylprednisolone is established only in young patients suffering from an isolated fracture of the spine.\n\nA number of current approaches to induce some regeneration of the spinal cord are on the way to becoming translated from the animal model to human beings. This is, indeed, an exciting area of research, as such approaches might lead to spinal cord repair. Unfortunately, the clinical significance of these approaches is frequently not critically questioned. Most of the agents have been studied in animal, usually rodent, models for more than 20 years and showed promising results. However, until now they have not yet been translated to humans or were not convincing in their effect3. A promising approach to inducing regeneration was the application of olfactory ensheathing cells (OECs). In a carefully conducted study, such autologous cells were transplanted to chronic, motor complete SCI patients without any beneficial effect14. What are the reasons for the problems in translating effects obtained in animal SCI models to the human condition? Some of them are listed below:\n\n- In the rodent model, the spinal cord is usually transected, while in humans after a trauma the spinal cord is damaged over two to three segments; this impedes sufficient meaningful regeneration\n\n- Several approaches were applied in complete human SCI (e.g. Nogo-Ab); however, in these patients, the remaining tissue bridges might not be sufficient to allow regeneration of tract fibres\n\n- In animals, agents are usually applied directly after spinal cord transection; in the human condition, this is usually not feasible earlier than 2 to 3 weeks after trauma (phase 1 Nogo-Ab study in our center) due to, e.g., surgery, complications such as infections of the urinary tract or pneumonia, and patient consent\n\n- Bridging was performed in chronic complete SCI patients (OEC transplantation); however, after 1 year, a neuronal dysfunction develops below the level of lesion that makes a success unlikely15,16\n\n- Cervical SCI patients would most benefit from some regeneration for their quality of life; however, in contrast to thoracic lesions, a cervical SCI is associated with damage (up to 40% of paresis17) of the peripheral nervous system (motoneurons and roots), and this makes the beneficial effects of regenerative substances and cells more unlikely\n\n\nConclusions\n\nIn the future, most probably, only by a combination of neuroprotective and neuroregenerative strategies can real progress in spinal cord repair be achieved. As a consequence, it should be pointed out that any substantial progress in managing SCI critically depends on a close cooperation among clinicians, engineers, and basic scientists.",
"appendix": "Competing interests\n\n\n\nThe author declares that he has no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAnderson KD: Targeting recovery: priorities of the spinal cord-injured population. J Neurotrauma. 2004; 21(10): 1371–83. PubMed Abstract | Publisher Full Text\n\nAhuja CS, Martin AR, Fehlings M: Recent advances in managing a spinal cord injury secondary to trauma. F1000. 2016; (in press).\n\nDietz V, Fouad K: Restoration of sensorimotor functions after spinal cord injury. Brain. 2014; 137(Pt 3): 654–67. PubMed Abstract | Publisher Full Text\n\nFehlings MG, Vaccaro A, Wilson JR, et al.: Early versus delayed decompression for traumatic cervical spinal cord injury: results of the Surgical Timing in Acute Spinal Cord Injury Study (STASCIS). PLoS One. 2012; 7(2): e32037. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nPhang I, Werndle MC, Saadoun S, et al.: Expansion duroplasty improves intraspinal pressure, spinal cord perfusion pressure, and vascular pressure reactivity index in patients with traumatic spinal cord injury: injured spinal cord pressure evaluation study. J Neurotrauma. 2015; 32(12): 865–74. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nWerndle MC, Saadoun S, Phang I, et al.: Monitoring of spinal cord perfusion pressure in acute spinal cord injury: initial findings of the injured spinal cord pressure evaluation study*. Crit Care Med. 2014; 42(3): 646–55. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDietz V, Müller R, Colombo G: Locomotor activity in spinal man: significance of afferent input from joint and load receptors. Brain. 2002; 125(Pt 12): 2626–34. PubMed Abstract | Publisher Full Text\n\nBarbeau H, Rossignol S: Enhancement of locomotor recovery following spinal cord injury. Curr Opin Neurol. 1994; 7(6): 517–24. PubMed Abstract | Publisher Full Text\n\nDietz V, Sinkjaer T: Spastic movement disorder: impaired reflex function and altered muscle mechanics. Lancet Neurol. 2007; 6(8): 725–33. PubMed Abstract | Publisher Full Text\n\nDietz V, Nef T, Rymer WZ: Neurorehabilitation Technology. London: Springer London; 2012. Publisher Full Text\n\nRiener R, Lünenburger L, Maier I, et al.: Locomotor Training in Subjects with Sensori-Motor Deficits: An Overview of the Robotic Gait Orthosis Lokomat. J Healthc Eng. 2010; 1(2): 197–216. Publisher Full Text\n\nBracken MB: Steroids for acute spinal cord injury. Cochrane Database Syst Rev. 2012; 1: CD001046. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBracken MB, Shepard MJ, Collins WF, et al.: A randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. Results of the Second National Acute Spinal Cord Injury Study. N Engl J Med. 1990; 322(20): 1405–11. PubMed Abstract | Publisher Full Text\n\nMackay-Sim A, Féron F, Cochrane J, et al.: Autologous olfactory ensheathing cell transplantation in human paraplegia: a 3-year clinical trial. Brain. 2008; 131(Pt 9): 2376–86. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDietz V, Grillner S, Trepp A, et al.: Changes in spinal reflex and locomotor activity after a complete spinal cord injury: a common mechanism? Brain. 2009; 132(Pt 8): 2196–205. PubMed Abstract | Publisher Full Text\n\nBeauparlant J, van den Brand R, Barraud Q, et al.: Undirected compensatory plasticity contributes to neuronal dysfunction after severe spinal cord injury. Brain. 2013; 136(Pt 11): 3347–61. PubMed Abstract | Publisher Full Text\n\nThomas CK, Zaidner EY, Calancie B, et al.: Muscle weakness, paralysis, and atrophy after human cervical spinal cord injury. Exp Neurol. 1997; 148(2): 414–23. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "14053",
"date": "27 May 2016",
"name": "Marios Papadopoulos",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14054",
"date": "27 May 2016",
"name": "Lorne Mendell",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1018
|
https://f1000research.com/articles/5-1017/v1
|
27 May 16
|
{
"type": "Review",
"title": "Recent advances in managing a spinal cord injury secondary to trauma",
"authors": [
"Christopher S. Ahuja",
"Allan R. Martin",
"Michael G Fehlings",
"Christopher S. Ahuja",
"Allan R. Martin"
],
"abstract": "Traumatic spinal cord injuries (SCIs) affect 1.3 million North Americans, producing devastating physical, social, and vocational impairment. Pathophysiologically, the initial mechanical trauma is followed by a significant secondary injury which includes local ischemia, pro-apoptotic signaling, release of cytotoxic factors, and inflammatory cell infiltration. Expedient delivery of medical and surgical care during this critical period can improve long-term functional outcomes, engendering the concept of “Time is Spine”. We emphasize the importance of expeditious care while outlining the initial clinical and radiographic assessment of patients. Key evidence-based early interventions (surgical decompression, blood pressure augmentation, and methylprednisolone) are also reviewed, including findings of the landmark Surgical Timing in Acute Spinal Cord Injury Study (STASCIS). We then describe other neuroprotective approaches on the edge of translation such as the sodium-channel blocker riluzole, the anti-inflammatory minocycline, and therapeutic hypothermia. We also review promising neuroregenerative therapies that are likely to influence management practices over the next decade including chondroitinase, Rho-ROCK pathway inhibition, and bioengineered strategies. The importance of emerging neural stem cell therapies to remyelinate denuded axons and regenerate neural circuits is also discussed. Finally, we outline future directions for research and patient care.",
"keywords": [
"Spinal cord injury",
"trauma",
"MRI",
"imaging",
"stem cell",
"neuroprotection",
"neuroregeneration"
],
"content": "Introduction\n\nTraumatic spinal cord injuries (SCIs) have devastating consequences for patients and families. Direct lifetime costs can be as high as $1.1–$4.6 million per patient, with over 1.3 million patients affected in North America alone1,2. Expedient delivery of specialized medical and surgical care can improve long-term functional outcomes for patients3,4. The rapidly evolving management of patients with SCI is summarized here with emphasis on evidence-based current practices and upcoming therapies in trial.\n\n\nPathophysiology\n\nThe initial primary trauma results in mechanical injury to cells, damages the sensitive microvasculature of the cord, and causes hemorrhage. Pro-apoptotic signaling is initiated and progressive edema contributes to ongoing ischemia5,6. Furthermore, the blood-spinal cord barrier is disrupted, permitting an influx of vasoactive peptides, cytokines, and inflammatory cells7,8. Over the ensuing hours to days, by-products of cellular necrosis are released (ATP, DNA, and K+), creating a cytotoxic post-injury milieu and activating microglia to further recruit phagocytes. Macrophages and polymorphonuclear leukocytes infiltrate and generate oxygen free radicals and other cytotoxic by-products. Excess glutamate release and failure of reuptake by astrocytes results in excitotoxicity for adjacent neurons9,10. Please see Figure 1.\n\n“(A) Primary and secondary mechanisms of injury determining the final extent of spinal cord damage. The primary injury event starts a pathobiological cascade of secondary injury mechanisms that unfold in different phases within seconds of the primary trauma and continuing for several weeks thereafter. (B) Longitudinal section of the spinal cord after injury. The epicenter of the injury progressively expands after the primary trauma as a consequence of secondary injury events. This expansion causes an increased region of tissue cavitation and, ultimately, worsened long-term outcomes. Within and adjacent to the injury epicenter are severed and demyelinated axons. The neuroprotective agents listed act to subvert specific secondary injuries and prevent neural damage, while the neuroregenerative agents act to promote axonal regrowth once damage has occurred. ATP = adenosine triphosphate.” Reprinted with permission from Wilson J, Forgione N, Fehlings MG. Emerging therapies for acute traumatic spinal cord injury. CMAJ. 2012; 185(6): 4854.\n\nAs the parenchymal volume is lost, cystic cavities coalesce, creating a physical barrier to cell migration11. Furthermore, the lack of structural framework impedes regenerative attempts. Over time, astrocytes proliferate and surround the perilesional zone, creating an irregular mesh-like barrier of interwoven cell processes12. This is accompanied by fibroblast deposition of chondroitin sulfate proteoglycans (CSPGs) including neural/glial antigen 2 (NG2) and tenascin13–16. CSPGs and myelin glycoproteins act via the Rho-ROCK (rho-associated protein kinase) pathway to inhibit neurite outgrowth by signaling growth cone collapse through effector kinases17. Together, these mechanisms severely restrict endogenous neural circuit regeneration and oligodendrocyte remyelination at a cellular level.\n\nSystemically, cervical and thoracic cord injuries can interrupt the sympathetic output of the intermediolateral column, causing neurogenic shock with loss of peripheral vascular tone and bradycardia18. The result can be profound hypotension, which further exacerbates cord ischemia19. Paralysis of the intercostal and abdominal muscles restricts the inspiratory phase of ventilation, leading to hypercarbia and/or hypoxia. Furthermore, a weakened cough, poor mobilization, and secondary immunodeficiency (immune paralysis) after SCI make patients highly susceptible to life-threatening infections20,21.\n\nThere is currently a lack of consensus on the optimal approach to several areas of SCI diagnosis and treatment, in part owing to heterogeneity in injuries (cervical versus thoracic, complete versus incomplete) but also owing to conflicting interpretations of the literature. As discussed below, early recognition and appropriate triage of patients are critical first-line components of care; however, the choice of imaging modalities for diagnosis and prognostication remains unclear22. Care is largely supportive, but the long-term importance of early surgical decompression (<24 hours)3,23,24, blood pressure augmentation (mean arterial pressure [MAP] ≥85 mmHg)4, and selective use of methylprednisolone (MPSS)25–28 is increasingly being recognized. Even under ideal circumstances, recovery of lost function is patient dependent and largely determined by their clinical status at 1–2 years post-injury. Below we summarize the current standards of care and discuss recent advances in the diagnosis, neuroprotection, prognostication, and regeneration for patients with SCI.\n\n\nCurrent care\n\nThe first-line care of a patient with SCI involves securing the airway, breathing, and circulation followed by appropriate spinal immobilization in the field to limit further insult of the highly susceptible cord during transport22. Recognition and appropriate triage of SCI patients is critical in the early period to ensure timely delivery of interventions at specialized centers22. While maintaining spinal immobilization, airway and breathing management can range from supplemental oxygen to intubation and ventilation. At any point during the acute injury, systemic hypotension (systolic blood pressure [SBP] <90 mmHg) is associated with worse neurologic outcomes22. With a profound loss of vascular tone and potential bradycardia, patients can rapidly fall into neurogenic shock. Large-volume intravenous (IV) fluid therapy (most often crystalloids) is the mainstay of treatment; however, adjunctive alpha-adrenergic vasopressors (e.g. norepinephrine and phenylephrine) may also be used to temporize patients. As soon as the patient is resuscitated, an American Spinal Injury Association (ASIA) International Standards for Neurological Classification of SCI (ISNCSCI) examination should be completed to establish the level of injury and baseline function22.\n\nEarly imaging to localize and classify the injury is critical to expeditiously manage patients and provide the outcome-altering early interventions discussed below3,4,23,24. CT is recommended in all patients, as plain radiographs can miss 6% of injuries29. In those with cervical injuries and high-energy mechanisms, imaging of the thoracolumbar spine is also recommended to detect concomitant injuries30. Magnetic resonance imaging (MRI) can be useful to assess ligamentous injury, critical disc herniations, and epidural hematomas and enhance prognostication of outcomes31; however, its role in the initial workup of patients with SCI remains unclear given resource constraints at many centers. Urgent MRI is recommended by the authors in cases with unexplained neurological deficits to ensure that ongoing cord compression or ligamentous injuries are not missed.\n\nWhile establishing the diagnosis and classifying the injury pattern, secondary injuries should be avoided by transferring the patient to an intensive care unit (ICU) setting with respiratory, hemodynamic, and cardiac monitoring22. Polytrauma patients should continue to have acute life- or limb-threatening injuries managed while maintaining appropriate spinal immobilization and recognizing early therapeutic windows for SCI interventions. This requires a concerted interdisciplinary effort including modified surgical positioning for orthopedic/general surgery procedures, fiberoptic tracheal intubation, and clear communication between teams.\n\nProgressive edema and hemorrhage contribute to ongoing mechanical pressure on the sensitive microvascular circulation. Surgical decompression aims to relieve this pressure to reduce secondary ischemic-hypoxic injury. The Surgical Timing in Acute Spinal Cord Injury Study (STASCIS) was a prospective, observational study of 313 patients with cervical SCI. Patients undergoing early decompression (<24 hours from injury; mean = 14 hours) were more than twice as likely to have a two-grade ASIA Impairment Scale (AIS) improvement at 6-month follow-up than those undergoing late surgery (≥24 hours from injury; mean = 48.3 hours)23. No difference in complication rates between early (24%) and late (30%) surgery was found (p = 0.21). These findings were further confirmed in a prospective Canadian cohort study24. Another observational study reported shorter hospital lengths of stay (LOS) for ASIA grade A (complete) or grade B (incomplete sensory injury; complete motor injury) patients undergoing early decompression3. An additional multi-center, European (SCI-POEM) study is currently underway31. The main critique of these studies is their cohort design, which was chosen for both practical reasons and ethical concerns surrounding randomizing patients where true clinical equipoise does not exist. However, these studies represent the most robust, large-scale data on surgery for SCI and provide support for a well-studied intervention in a field where few treatment options exist for patients. Given this data, the concept of “Time is Spine” has emerged, emphasizing the critical importance of early therapies to improve long-term functional outcomes4. Furthermore, early surgical decompression (<24 hours) is recommended in current American Association of Neurological Surgeons (AANS) and Congress of Neurological Surgeons (CNS) guidelines22.\n\nTo further mitigate ischemia of the injured cord, blood pressure augmentation has emerged as a viable strategy. Maintaining MAP ≥85–90 mmHg post-injury has been shown to improve AIS grade outcomes for patients4. Current AANS/CNS guidelines provide level III recommendations to maintain MAP for 7 days post-injury. This requires maintenance of a euvolemic or slightly hypervolemic state using IV crystalloid in addition to an infusion of vasopressors and invasive blood pressure monitoring (e.g. arterial line). These significant requirements have led to a non-inferiority trial of MAP ≥65 mmHg versus MAP ≥85 mmHg called MAPS, which is expected to be completed in 201731.\n\nMaintaining these MAP parameters can also be a barrier to mobilization, which is critical in the early post-injury period to prevent complications. Safely elevating patients and engaging muscle activity requires a collaborative, multidisciplinary effort along with adjuncts such as prophylactic vasopressors and peripheral/abdominal binding. The ideal time to begin mobilization should be evaluated on an individual basis according to the patient’s hemodynamics, underlying comorbidities, and the expertise of the healthcare team.\n\nMPSS is a synthetic corticosteroid which upregulates anti-inflammatory factors and decreases oxidative stress to enhance endogenous cell survival in animal models of SCI. A series of key clinical trials entitled National Spinal Cord Injury Study (NASCIS) I (1984)25, II (1990)26, and III (1997)27 demonstrated serious adverse events with a high-dose MPSS protocol (e.g. sepsis), which outweighed the potential benefit for neurologic recovery. However, when a low-dose protocol was given to patients within 8 hours of injury, no adverse events and a potential improvement were seen. The study methodology and subgroup analyses from this series have been extensively debated over the last several decades. To settle this debate, a comprehensive Cochrane review was published in 2012 encompassing six randomized controlled trials (RCTs) and several key observational studies. The analysis demonstrated a four-point ASIA motor score improvement for patients receiving MPSS within 8 hours of injury33. While this appears to be a small improvement in relative terms, a four-point improvement in key myotomes such as grip, triceps, and deltoid function can represent a significant functional gain for patients. The 2016 AOSpine guidelines, developed by an international and interdisciplinary committee of experts, will suggest IV MPSS (administered over 24 hours) as a treatment option when feasible to patients within 8 hours of injury.\n\n\nFrontiers of imaging\n\nConventional MRI, producing T1- and T2-weighted images, has been shown to be of modest value in helping to predict neurological and functional outcomes, particularly the prognostic factors of length of hemorrhage and degree of cord compression31. However, conventional MRI fails to yield information about the health of the spinal cord tissue as signal changes are non-specific and can reflect a range of physiological processes such as hemorrhage (macroscopic or microscopic), edema, gliosis, cell loss, and cavitation31. A number of emerging MRI techniques have the potential to substantially improve our ability for prognostication by quantifying the degree of tissue injury and measuring functional changes within the spinal cord34. Techniques that can quantify aspects of tissue microstructure include diffusion tensor imaging (DTI), reflecting axonal integrity, magnetization transfer (MT) and myelin water fraction (MWF), correlating with myelin quantity, and MR spectroscopy (MRS), measuring the concentration of key molecules that reflect cell loss (N-acetylaspartate), gliosis (myo-inositol), and ischemia (lactate). Functional MRI (fMRI) can visualize neuronal activity and connectivity. All of these techniques are under intense investigation, with DTI in particular showing strong correlation with tissue injury, which may lead to clinical translation in the near future35.\n\n\nFrontiers of neuroprotection\n\nNeuroprotective interventions to preserve injured tissue and reduce secondary insult are key approaches in SCI. Multiple therapies targeting components of the pathophysiologic cascade are currently under investigation and in trial.\n\nHypothermia decreases the basal metabolic rate of central nervous system tissue and reduces inflammation36. Therapeutic hypothermia (32–34°C) has been applied in the neuroprotection of patients after cardiac arrest37 and neonatal hypoxic-ischemic encephalopathy38,39. Animal models of SCI have demonstrated significant improvements with systemic intravascular cooling40, leading to a pilot study of 14 AIS grade A patients in which a trend towards neurologic improvement (43% versus 21%) was reported with no difference in complication rates41. The pending Acute Rapid Cooling Therapy for Injuries of the Spinal Cord (ARCTIC) phase II/III trial looks to further assess the efficacy of this therapy42.\n\nRiluzole is a benzothiazole, voltage-gated sodium-channel blocker which indirectly decreases glutamate release and enhances reuptake43. It has been used successfully to slow the progression of motor neuron loss and improve survival in patients with amyotrophic lateral sclerosis44. A phase I/II clinical trial of 36 patients with SCI demonstrated 15.5-point improvements in ISNCSCI motor scores for riluzole-treated patients with a cervical level injury45. A phase II/III RCT entitled “Riluzole in Spinal Cord Injury Study” (RISCIS), sponsored by AOSpine, the North American Clinical Trials Network (NACTN), the Rick Hansen Institute, and the Ontario Neurotrauma Foundation, is now underway to further assess efficacy for patients with C4-8 level injuries. The trial is expected to complete in 201832.\n\nMinocycline is a tetracycline-class antibiotic with anti-inflammatory properties including inhibition of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), nitric oxide synthase (NOS), and microglial activation. Preclinical models of SCI showed dramatically decreased lesion sizes and neuron loss with acute minocycline treatment46,47. In a mixed-level phase II study, cervical SCI patients (N = 25) had a 14-point ASIA motor score improvement with minocycline treatment versus placebo (p = 0.05)48. This has led to a phase III trial (N = 248) of IV minocycline x 7 days versus placebo entitled “Minocycline in Acute Spinal Cord Injury” (MASC) to be completed by 201832.\n\nFibroblast growth factor (FGF) is part of the family of heparin-binding proteins. It has been shown to protect against excitotoxic cell death and mitigate oxygen free radical production in animal models of SCI49. SUN13837 (Asubio Pharmaceuticals Inc.) is an FGF analogue trialed in a phase I/II study which completed in 2015. Results are expected to be reported in the near future32. Similarly, cytokine granulocyte-colony stimulating factor (G-CSF) has been shown to be neuroprotective in SCI by directly promoting cell survival and inhibiting TNF-α and IL-1β50. Two non-randomized studies demonstrated improvements in AIS scores for patients receiving IV G-CSF51,52. A larger randomized trial is anticipated.\n\nFinally, magnesium is a non-competitive NMDA receptor antagonist. It has been applied in the neuroprotection of multiple central nervous system disorders to decrease excitotoxicity and inhibit inflammation. When delivered with an excipient, such as polyethylene glycol (PEG), it generates stable cerebrospinal fluid levels in the therapeutic range53–55. AC105 (Acorda Therapeutics) is a Mg-PEG compound that was studied in a phase I trial concluding in February 201532. Results are pending report.\n\n\nFrontiers of neuroregeneration\n\nThe majority of patients living with impairments from SCI are in the chronic phase of injury. Neuroregenerative strategies aiming to help these millions of patients are being developed by countless researchers worldwide. Significant therapeutic opportunities exist using endogenous and exogenous repair mechanisms with adjuncts to address barriers to recovery such as the loss of structural framework, cystic cavitation, astroglial/CSPG scarring, and inhibitory molecular signaling.\n\nCSPGs, myelin-associated glycoproteins (MAGs), oligodendrocyte-myelin glycoprotein (OMgp), and neurite outgrowth inhibitor-A (NOGO-A) all act on receptors associated with the Rho-ROCK pathway to inhibit neurite outgrowth, thereby stemming attempts at recovery. Multiple types of drugs directed at disrupting this signaling cascade have been developed. Bioengineered monoclonal NOGO-A antibodies, given by intrathecal injection, have been shown to improve regeneration of rat and primate spinal cords56,57. A phase I study (N = 51) of ATI355 (anti-Nogo-A antibody) has been completed with results pending dissemination32. Direct Rho inhibition has also been developed in the form of an intraoperatively applied epidural paste (Cethrin/VX-210; Vertex Pharmaceuticals)17. A mixed cervicothoracic-level phase I/IIa study (N = 48) demonstrated significant motor improvement (18.5 ASIA motor score points) for cervical patients receiving Cethrin without any increase in complications58. A further phase IIb trial is planned.\n\nInstead of inhibiting the Rho-ROCK pathway, chondroitinase ABC (ChABC) is an enzyme which degrades CSPGs in the glial scar to effectively remove initiators of the cascade. In rodent models of SCI, intrathecal and intraparenchymal treatments with ChABC have been shown to reduce CSPGs, scar volume, and cavity volume. Electrophysiologic and behavioral improvements in motor and sensory function after ChABC treatment have also been demonstrated by a number of groups including seminal work by Bradbury et al.59–61. This exciting approach is being further developed with novel delivery methods and in combination with other regenerative techniques such as cell-based therapy49,60,62. Furthermore, a humanized form of chondroitinase is being studied with a more central nervous system-specific motif.\n\nCell-based therapies are a rapidly evolving field of regenerative medicine. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and their differentiated progeny, are capable of regenerating lost neural circuits, remyelinating denuded axons, modulating the inflammatory response, and modifying the microenvironment63–65. ESCs have been studied the longest but are in limited supply and their use raises complex ethical issues. The discovery of four factors capable of generating a pluripotent cell from adult somatic cells provided a limitless source of cells with the possibility of developing autologous therapies in the future66. While previously unknown issues with iPSCs, such as epigenetic memory and early senescence, are being studied, these cells remain a key therapeutic strategy67. Multiple studies of oligodendrocyte precursor cells, neural precursor cells, and cells to modify the microenvironment have produced recovery of function in preclinical models over the past three decades68–73. An international phase I/II trial of human central nervous system stem cell injections for cervical SCI is being conducted by Stem Cells Inc. with results expected in 201732. A parallel thoracic injury phase I/II study, currently completing follow-ups, has shown improvements in sensation with no increase in complication rates74. Another phase I trial of NSI-566 neural stem cells for thoracic injury is expected to conclude in 201632. Ongoing studies will continue to address safety concerns and establish efficacy of this exciting therapy.\n\nSeveral important parallel cell-based strategies are under investigation. Schwann cells (SCs) are able to remyelinate both peripheral nervous system (PNS) and central nervous system axons and are a key component of effective PNS regeneration. In animal models, SCs have been shown to reduce cystic cavitation, enhance tissue sparing, and promote behavioral recovery75. The Miami Project to Cure Paralysis is currently recruiting patients with chronic ASIA grade A, B, and C cervical and thoracic injuries for a phase I (N = 10; NCT02354625), open-label trial of autologous SCs transplanted into the injury epicenter32. The study is expected to conclude in 2018. The same team is also running a phase I study (N = 10; NCT01739023) of autologous SCs for subacute thoracic ASIA grade A SCI expected to report in 201632.\n\nOlfactory ensheathing cells (OECs) cover olfactory neurons in a manner similar to SCs. They are potent phagocytes capable of continuously clearing microbes and debris from the nasal mucosa while also secreting neurotrophic support factors76–79. OECs harvested from the nasal mucosa or olfactory bulb have been shown to enhance axonal regeneration and remyelination and significantly improve behavioral outcomes in animal models80. Several chronic SCI trials of OECs have been completed and compiled in a recent meta-analysis (cumulative N = 1193) which demonstrated no significant increase in serious adverse events. Higher-quality studies are required moving forward to definitively establish efficacy81.\n\nMesenchymal stem cells (MSCs) are multipotent stromal cells with the capacity to repair damaged tissues by differentiating along connective tissue lineages (e.g. chondrocytes, myocytes, osteoblasts, and adipocytes)82. Furthermore, they are uniquely capable of modulating the inflammatory response both at a systemic level and within their local environment83–85. In animal models, MSCs have been shown to decrease peripheral inflammatory cell infiltration, enhance pro-survival trophic factor levels, and promote neural tissue sparing86,87. Numerous phase I and II trials studying autologous MSCs are ongoing worldwide. Pharmicell Co. is conducting a phase II/III trial (N = 32; NCT01676441) of autologous MSCs transplanted into the parenchyma and intrathecal space of patients with ASIA grade B injuries. The study is expected to conclude in 201632. A similar class of support cells is bone marrow cells (BMCs) which, in preclinical testing, have been shown to facilitate directed axonal regrowth by producing extracellular matrix88 and promoting remyelination89. A phase I/II RCT (N = 21) of ASIA grade A patients administered autologous BMCs intraparenchymally or intrathecally was published in 2015. No serious adverse events were reported90. A similar recent study in children with chronic SCI also showed no significant adverse events91. Bioengineered strategies form an important complementary avenue of research for regeneration of the traumatically injured cord. Multiple biomaterials have been developed to fill cavitation defects and recreate the structural architecture required to promote endogenous and exogenous cell migration and survival92–96. These materials are being engineered to have a specified porosity and density, be immunologically inert, and biodegrade over time. Furthermore, many have been modified to release growth factors or immunomodulatory drugs to enhance regeneration95,97,98. A unique class of biomaterial, self-assembling peptide hydrogels, has been designed to be injectable and assemble into nanofibrils resembling extracellular matrix when exposed to ionic or temperature changes68,99. As biochemical manufacturing and our transplant techniques are refined, biomaterials are likely to be important components of a successful regenerative therapy for SCI.\n\n\nFrontiers of rehabilitation\n\nA critical part of any treatment for SCI is an effective rehabilitation strategy. This requires the integration of SCI-specific physiotherapy (e.g. stretching, strength training, and transferring), occupational therapy (e.g. modified activities for self-care), nursing (e.g. wound care and bowel/bladder care), psychology, speech-language pathology, and medicine. Conventional physical rehabilitation aims to reduce chronic complications (e.g. ulcers, deformity, and cardiorespiratory deconditioning) while enhancing residual function for maximal gain. Several technological adjuncts are actively being researched and integrated into long-term rehabilitation to achieve these goals including functional electrical stimulation (FES) and epidural stimulation (EDS). FES applies microcurrents to nerves and muscles to enhance motor function during rehabilitation or daily activities. Patterned FES has shown success in restoring both upper extremity (e.g. writing, eating, and self-care) and lower extremity (e.g. supported ambulation and stationary bicycle riding) function. FES has also been used to significantly improve volitional control of the bowel and bladder100. In addition to immediate gains, FES may also produce long-term improvements similar to activity-based restorative therapy (ABRT) via mechanisms of neuroplasticity. Both ABRT and FES repeatedly activate preserved circuits to maintain existing connections while promoting synaptogenesis, myelination, and neurite sprouting100–103. Furthermore, during physical rehabilitation, FES augmentation can dramatically increase patients’ oxygen uptake and respiratory rate and improve their fat to muscle ratio104,105. A phase III trial (N = 84; NCT01292811) of FES for the restoration of upper limb function in tetraplegic patients with subacute cervical injuries is currently recruiting patients. This study is expected to conclude in 2018. EDS is a parallel approach using microcurrents delivered by epidural electrodes to stimulate the spinal cord and/or conus medullaris106. It has been successfully used in the treatment of refractory neuropathic pain for numerous conditions (e.g. amputation, stroke, and SCI). The concept behind EDS-induced motor recovery is the enhancement of neuroplasticity by activating central circuits including the central pattern generator for locomotion (T11-L1) and cardiorespiratory circuits. Several phase I and II studies (NCT02592668, NCT02339233, and NCT02313194) are underway to explore the potential of EDS with results expected over the next 5 years32.\n\n\nLooking forward\n\nThe landscape of SCI management is quickly changing as the heterogeneity of patients and long-term importance of key early interventions are increasingly being recognized. Combinatorial neuroprotective and neuroregenerative strategies are most likely to be effective moving forward given the multifaceted nature of the injury; however, this approach may require tailoring to specific patient subgroups. This necessitates a deeper understanding of SCI pathophysiology, clinical presentation, and relevant imaging, serum, and cerebrospinal fluid biomarkers107,108. While landmark studies of the past have enrolled varied groups of patients for logistical reasons, we foresee future studies stratifying patients by well-defined diagnostic criteria to elucidate subtle but prognostically important differences. The results of the above trials may become catalysts for critical changes in the current standard of care. Even small improvements in sensory or motor outcomes can have profound functional effects on patients’ vocational abilities and independence.\n\n\nAbbreviations\n\nAANS, American Association of Neurological Surgeons; ABRT, activity-based restorative therapy; AIS, American Spinal Injury Association Impairment Scale; ASIA, American Spinal Injury Association; BMC, bone marrow cell; ChABC, chondroitinase ABC; CNS, Congress of Neurological Surgeons; CSPG, chondroitin sulfate proteoglycan; DTI, detrusor tensor imaging; EDS, epidural stimulation; ESC, embryonic stem cell; FES, functional electrical stimulation; FGF, fibroblast growth factor; G-CSF, granulocyte-colony stimulating factor; IL-1β, interleukin-1β; iPSC, induced pluripotent stem cell; ISNCSCI, International Standards for Neurological Classification of Spinal Cord Injury; IV, intravenous; MAP, mean arterial pressure; MPSS, methylprednisolone; MRI, magnetic resonance imaging; MSC, mesenchymal stem cell; NOGO-A, neurite outgrowth inhibitor-A; OEC, olfactory ensheathing cell; PEG, polyethylene glycol; PNS, peripheral nervous system; RCT, randomized controlled trial; ROCK, rho-associated protein kinase; SBP, systolic blood pressure; SC, Schwann cell; SCI, spinal cord injury; STASCIS, Surgical Timing in Acute Spinal Cord Injury Study; TNF-α, tumor necrosis factor-α.",
"appendix": "Competing interests\n\n\n\nMichael Fehlings is an investigator in the Stem Cells Inc. trial mentioned in this article. The authors have no other conflicts or potential conflicts to disclose.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThank you to Madeleine O’Higgins for manuscript copyediting.\n\n\nReferences\n\nNational Spinal Cord Injury Statistical Center: Spinal cord injury facts and figures at a glance. J Spinal Cord Med. 2014; 37(1): 117–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFoundation CaDR: One degree of separation: paralysis and spinal cord injury in the United States. 2010. Reference Source\n\nDvorak MF, Noonan VK, Fallah N, et al.: The influence of time from injury to surgery on motor recovery and length of hospital stay in acute traumatic spinal cord injury: an observational Canadian cohort study. J Neurotrauma. 2015; 32(9): 645–54. 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Biomaterials. 2013; 34(15): 3775–83. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nTam RY, Cooke MJ, Shoichet MS: A covalently modified hydrogel blend of hyaluronan–methyl cellulose with peptides and growth factors influences neural stem/progenitor cell fate. J Mater Chem. 2012; 22(37): 19402–19411. Publisher Full Text | Faculty Opinions Recommendation\n\nAnsorena E, De Berdt P, Ucakar B, et al.: Injectable alginate hydrogel loaded with GDNF promotes functional recovery in a hemisection model of spinal cord injury. Int J Pharm. 2013; 455(1–2): 148–58. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nItosaka H, Kuroda S, Shichinohe H, et al.: Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering. Neuropathology. 2009; 29(3): 248–57. PubMed Abstract | Publisher Full Text\n\nTaylor SJ, McDonald JW 3rd, Sakiyama-Elbert SE: Controlled release of neurotrophin-3 from fibrin gels for spinal cord injury. J Control Release. 2004; 98(2): 281–94. PubMed Abstract | Publisher Full Text\n\nVulic K, Shoichet MS: Tunable growth factor delivery from injectable hydrogels for tissue engineering. J Am Chem Soc. 2012; 134(2): 882–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu Y, Ye H, Satkunendrarajah K, et al.: A self-assembling peptide reduces glial scarring, attenuates post-traumatic inflammation and promotes neurological recovery following spinal cord injury. Acta Biomater. 2013; 9(9): 8075–88. PubMed Abstract | Publisher Full Text\n\nMartin R, Sadowsky C, Obst K, et al.: Functional electrical stimulation in spinal cord injury:: from theory to practice. Top Spinal Cord Inj Rehabil. 2012; 18(1): 28–33. PubMed Abstract | Publisher Full Text\n\nLavrov I, Gerasimenko YP, Ichiyama RM, et al.: Plasticity of spinal cord reflexes after a complete transection in adult rats: relationship to stepping ability. J Neurophysiol. 2006; 96(4): 1699–710. PubMed Abstract | Publisher Full Text\n\nCourtine G, Gerasimenko Y, van den Brand R, et al.: Transformation of nonfunctional spinal circuits into functional states after the loss of brain input. Nat Neurosci. 2009; 12(10): 1333–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDietz V, Harkema SJ: Locomotor activity in spinal cord-injured persons. J Appl Physiol (1985). 2004; 96(5): 1954–60. PubMed Abstract | Publisher Full Text\n\nBhambhani Y, Tuchak C, Burnham R, et al.: Quadriceps muscle deoxygenation during functional electrical stimulation in adults with spinal cord injury. Spinal Cord. 2000; 38(10): 630–8. PubMed Abstract\n\nKakebeeke TH, Hofer PJ, Frotzler A, et al.: Training and detraining of a tetraplegic subject: high-volume FES cycle training. Am J Phys Med Rehabil. 2008; 87(1): 56–64. PubMed Abstract | Publisher Full Text\n\nHarkema S, Gerasimenko Y, Hodes J, et al.: Effect of epidural stimulation of the lumbosacral spinal cord on voluntary movement, standing, and assisted stepping after motor complete paraplegia: a case study. Lancet. 2009; 377(9781): 1938–47. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPouw MH, Hosman AJ, van Middendorp JJ, et al.: Biomarkers in spinal cord injury. Spinal Cord. 2009; 47(7): 519–25. PubMed Abstract | Publisher Full Text\n\nCadotte DW, Fehlings MG: Will imaging biomarkers transform spinal cord injury trials? Lancet Neurol. 2013; 12(9): 843–4. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "14055",
"date": "27 May 2016",
"name": "Lorne Mendell",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14056",
"date": "27 May 2016",
"name": "Angela Gall",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1017
|
https://f1000research.com/articles/5-1015/v1
|
27 May 16
|
{
"type": "Review",
"title": "Some vexations that challenge viral immunology",
"authors": [
"Barry T. Rouse",
"Scott N. Mueller"
],
"abstract": "The field of viral immunology seeks to understand mechanisms of virus-host interaction with a view of applying this knowledge to the design of effective vaccines and immunomodulators that control viral infections. This brief review discusses several areas of the field that hold substantial promise for translation, but where further work is critically required to find solutions. We emphasize that our fundamental understanding of virus-host relationships is moving in leaps and bounds, but we lag behind in applying this knowledge to the successful control of many viral infections.",
"keywords": [
"viral immunology",
"vaccine",
"immunomodulators",
"immunology",
"acquired immunity"
],
"content": "Introduction\n\nThe science of immunology has its roots in infectious disease, with its pioneers striving to explain how a body defends itself against infections and why vaccines protect in many, although not in all, instances. Few immunologists can claim to have applied the fundamental understanding of their science to develop effective new vaccines, but this has always been the expectation. In fact, the victory of vaccines (almost all developed empirically) is complete for some agents but is notably absent for many others. Immunologists might believe that once all the mechanistic facts are in and digested, it is just a matter of time before strategies will be designed to deal with current vexations in infectious disease, autoimmunity, allergy and asthma, transplantation, cancer, and even subtle problems that affect the nervous system. This optimism is based on the enormous accumulation of data, the availability of many powerful in vivo models and in vitro test systems, the enthusiasm, confidence, and energy of researchers, and, yes, the abundance of funding from both government sources and private philanthropists. However, translation of such abundant and complex experimental data into a sufficiently deep understanding of the biology of the immune system that will permit the design of effective vaccines against what are currently elusive targets (i.e. HIV) is a goal we are yet to reach. In this brief review, we discuss some topics in the field of viral immunology that remain problematic but could be resolved as a consequence of accumulating new data and ideas. We expect to encourage argument and hope to inspire solutions.\n\n\nHow should we deal with viruses that lack effective vaccines?\n\nThis question becomes more pressing with agents that persist and cause chronic lesions, but new pathogens are always emerging (for example Ebola, or in recent years the Zika virus). Moreover, several commonly occurring acute viral infections also lack effective vaccines. A significant example is respiratory syncytial virus (RSV), a frequent cause of respiratory disease in infants, especially those born prematurely. Vaccination in early childhood is often not successful due to poor adaptive immunity and Th2-skewed responses. Progress in vaccine development for RSV has been slow for several reasons. A vaccine produced several years ago proved highly unsatisfactory, since this inactivated vaccine caused some recipients to express enhanced disease when exposed to natural infection, likely because the vaccinees developed immunopathological reactions1. Other reasons for slow vaccine development include the lack of ideal animal models to study RSV pathogenesis and the fact that natural infection of infants does not make them immune to reinfection because of their immature immune systems. However, hope for success recently came from structural studies on the F protein, a target for neutralizing antibodies needed to protect against RSV1. It was shown that neutralizing antibody-inducing immunogenic epitopes could be expressed on the RSV F protein if it was reengineered to prevent the loss of neutralizing antibody stimulating epitope expression, as happens normally when the native F protein fuses with the cell the virus infects1. We anticipate that structural biologists might provide insights about ligand-antibody complexes for other viral proteins, such as with influenza and HIV, and this will result in the design of immunoprotective vaccine formulations. This topic was recently reviewed2.\n\nWith regard to persistent pathogens, two of the most troublesome chronic viral infections that lack vaccines are HIV and hepatitis C virus (HCV). Fortunately, both viruses can be effectively controlled by combination drug therapy, but this is not an ideal solution and is prohibitively expensive in the case of HCV. For these two diseases, prophylactic as well as therapeutic vaccines are needed. Other chronic infections, such as hepatitis B virus (HBV) and human papilloma viruses (HPVs), do have effective prophylactic vaccines, but there is a need for therapeutic vaccines for those already infected3. However, producing effective therapeutic vaccines against any infection provides a major challenge.\n\nThe search for a vaccine against HIV has been vigorously pursued since the virus was first identified in the early 1980s. We have experienced periodic occasions of apparent success4, but none have withstood the scrutiny of independent verification. However, some evidence inspires optimism that an effective vaccine could eventually be produced. It is well known that some infected persons who are not receiving therapy successfully control the infection for prolonged periods of time5. Such elite controllers indicate that a protective immune response can occur, although defining the immunological signature of control and, importantly, duplicating it with a vaccine has proven elusive. Moreover, elite controllers do not eliminate the virus from their system and some patients ultimately lose elite status likely because of the eventual emergence of virus variants that manage to escape the determined efforts of the immune system.\n\nOptimism for an eventual effective HIV vaccine comes from animal model studies on lentivirus infections in primates6. Many different approaches have been explored and shown promise, but none more so than that pioneered by Louis Picker and colleagues7. This group showed that engrafting selected simian immunodeficiency virus (SIV) proteins into a rhesus cytomegalovirus gene modified vector (Rh-CMV) induced a high level of protective immunity which in many cases fully controlled virus replication upon challenge with a highly virulent SHIV challenge7. The success of this form of immunization was likely explained by a very broadly reactive and unusual CD8+ T cell response that was largely composed of CD8+ T cells that recognize peptides presented by class II major histocompatibility complexes (MHCs) rather than the normal class I MHC restricting elements. These CD8+ class II restricted responses were unique and did not overlap with the conventional CD8+ responses. The latter dominate when animals were vaccinated with other types of vaccines. The Picker results are highly encouraging and were duplicated using Rh-CMV vectors containing some other antigens, most notably Mycobacterium tuberculosis (not yet published). One hopes the Picker approach is independently confirmed and can be tested to see if a similar protective pattern of responsiveness can be achieved against HIV in humans. Also in need of explanation will be the observation that the Rh-CMV vector vaccination approach was successful in only approximately 50% of animals, yet all developed the predominant non-canonical pattern of CD8+ T cell responsiveness.\n\nWhereas several viral infections are still in need of successful prophylactic vaccines, in many instances we also require therapeutic vaccines that could boost inadequate immunity. This is a requirement in many chronic infections, which includes almost all herpesvirus infections as well as HIV, HCV, and HBV and those chronically infected with HPV who missed out on the highly effective prophylactic vaccines. There are only a few success stories with therapeutic vaccines, but the one which stands out is the apparent success of varicella zoster vaccine used to diminish the chance of shingles in elderly patients infected decades previously with the chickenpox virus8. However, the need for effective therapeutic vaccines is emphasized in the case of HCV infection. In this instance, 20–30% of persons infected with HCV successfully resolve their infection and usually remain immune to reinfection9. Unfortunately, the majority fail to resolve their infections and these persons undergo gradual deterioration in liver function, which may ultimately fail, or worse still evolve into hepatocellular carcinoma. In the affluent world, chronic HCV can now be controlled using an inexcusably expensive new drug combination therapy10. Nevertheless, once the virus is cleared in those fortunate drug recipients, they remain fully susceptible to reinfection, which in fact often occurs10. Thus, with HCV we need therapeutic vaccines that would protect not only those who are chronically infected but also patients who have undergone a successful form of drug therapy. These issues lead to the general question of whether the balance of immunity can be changed in those situations where it is ineffective or even contributes to tissue damage.\n\n\nManipulating memory responses\n\nA hallmark of acquired immunity is memory, a topic that interests all viral immunologists, especially those striving to improve vaccines or control infections in other ways. Many rules for memory have been established using mouse models, some of which apply and others which may differ somewhat in humans. Memory involves lymphocytes and their derivatives and once a pattern is established, it may be hard to change; certainly in humans, memory can be very long lasting. It is becoming increasingly evident that the components of memory reside in different locations, which in some instances are resistant to relocation11. Moreover, in adult humans, the great majority of memory cells are located at mucosal, skin, or non-lymphoid tissue sites. Indeed, it is now evident that the memory cells most instrumental in providing the first line of protection against pathogens are located at the tissue and mucosal entry sites. We now have a better understanding of how these tissue-resident memory (TRM) cells get there and that a genetic program directed in part by the tissues restricts the expression of molecules that would promote their relocation11,12. However, we need to better understand how TRM cells are regulated in tissues, how they contribute to protective immunity, how long they remain immunoprotective, and if they can be expanded in numbers and function. Clues are emerging on all fronts and the topic has received lucid reviews11,12. It seems evident that immunity to reinfection, even in those already infected, as occurs with many viruses (particularly RNA viruses but several herpes viruses too), might depend critically on the numbers and function of TRM cells. This was well established with HSV in mice13, but the main practical question is how we can expand and boost local memory in humans. Artificially, this can be achieved in mice by pulling cells into tissue sites by the injection of relevant chemokines14. Another approach might be to cause TRM cells to multiply and become more functional in situ perhaps by using appropriate innate immune ligands with or without cocktails of cytokines, such as interleukin (IL)-7 and IL-15. Although the maintenance of TRM cells in mice does not require antigen, it remains possible that the provision of some form of antigen and/or co-stimulators might succeed in expanding TRM populations, presumably via recruitment of more cells into the tissue. However, we need to remain cautious, since recruiting very large numbers of T cells into the tissues could have detrimental effects. These issues are of high importance and are under active investigation by many groups, which includes one of us.\n\nAnother important aspect that impacts on immune protection is its potential erosion during persistent infections, commonly referred to as exhaustion15. T cells constantly exposed to antigen show a multi-faceted change to their differentiation state, including a progressive diminishment of effector functions, upregulation of a series of inhibitory receptors (including PD-1, TIM-3, LAG-3, CTLA-4, and others), and altered gene expression and metabolism. This dramatic change in T cell fate negatively impacts memory T cell generation. First characterized during chronic LCMV infection in mice, many persistent human infections and cancers induce this dysfunctional state in responding T cells. Of notable interest for vaccines and therapies, impeding ligand binding to these receptors causes a rebound of T cell activity and better control of infection15. This inhibitor blocking approach, along with the manipulation of other checkpoint controls on T cell function, has found exciting application to the control of some cancers16. Yet it remains to be seen whether such checkpoint blockade could improve outcomes in patients with established chronic infections such as HIV and HCV. Encouragingly, combining checkpoint blockade with therapeutic vaccination to enhance T cell responses and improve viral control has shown promise in models of both HPV and SIV infection3,17. Indeed, improving T cell functions may help improve therapeutic vaccine efficacy against HCV and perhaps even against herpesviruses.\n\n\nBlunting immune-mediated tissue damage\n\nIn many virus-induced lesions, the tissue damage results from immunopathology and this is the consequence of an imbalanced response to infection18. Accordingly, control could result if the balance of the response was reprogrammed, although this objective is more feasible during the induction compared to the effector phase of immunity. With regard to rebalancing the pattern of adaptive immunity, several approaches show promise, although most are not antigen specific and hence could have unwanted side effects. The non-specific approaches include using modulators of innate immunity that impact on the pattern of adaptive immune responsiveness19, manipulating the expression of host molecules that put brakes on immune protection, such as galectins and lipid-derived mediators20, or exploiting metabolic differences between cell subsets to selectively expand protective T cells21. Our own work has focused on manipulating the levels and functions of regulatory T (Treg) cells in virus-induced tissue damaging lesions, as this approach could conceivably be made antigen specific. Manipulating the T-effector/Treg cell balance is most easily achieved during the induction phase, but the usual clinical demand is to reformulate the pattern of events once an unfavorable profile has been established. A clue for the potential success of reformulating profiles may be the discovery that the function of fully differentiated T cell subsets is plastic and can be reprogrammed in response to ongoing events in their environment22. The focus has been on plasticity changes between pro-inflammatory cells such as Th1 and Th17 CD4+ T cells in relation to numbers of Treg cells. Understanding and harnessing plasticity to manage immune-mediated autoimmune disease has been championed by the Bluestone group22. In viral-induced immunoinflammatory disease, we usually need to expand Treg cells at the expense of pro-inflammatory T cells. Few practical avenues are available as yet to accomplish reprogramming of fully differentiated T cells in vivo, but those approaches which do seem promising include the targeting of cytokines such as IL-6, IL-1, and IL-12 that are involved in driving Treg plasticity22. Since plasticity involves changes in epigenetic control, this process can be targeted with drugs that block DNA methyltransferases23 or histone deacetylases24. Other potentially practical approaches are to target downstream signaling events which differ between Treg and T-effector cells. This can be achieved with the drug rapamycin, which inhibits mTORC1. In T effectors, mTORC1 is involved in the production of inflammatory cytokines which will be inhibited by rapamycin. In Treg cells, mTORC1 is not active except when the cells are undergoing plasticity. Hence, rapamycin will not inhibit functional Treg cells and as a bonus will stop the cells from becoming proinflammatory. The outcome will be a change in the overall balance that favors Treg cells22. Perhaps of particular value eventually will be to exploit the known differences in glucose and fatty acid metabolism by Treg and effector T cells21. For example, Treg cells can be expanded based upon their known requirements of high fatty acid oxidation and low glucose consumption. This can be achieved with drugs such as soraphen A, which inhibits fatty acid synthesis25, and 2-deoxyglucose, which blocks glycolysis26.\n\n\nHow can novel technology lead to making better vaccines?\n\nVaccines are among the greatest success stories in modern medicine, but in most cases we do not fully understand how host defense mechanisms account for their success. Such information is necessary, since it should help guide vaccine improvements and could reveal how to develop vaccines against the many agents that still lack them. The information needed to design novel effective vaccines is expected to come from the tools elaborated by systems biology, popularly referred to as “omics”27. These approaches are all high throughput and assemble an abundance of data, which include DNA microarrays, proteomics, genomics, transcriptomics, metabolomics, epigenomics, and deep sequencing. The expectation is that this combination of high-throughput data, along with measurements of immunological parameters by conventional immunological assays, could be subjected to complex computational analysis to yield a signature indicative of optimal immunogenicity and vaccine efficacy or failure. Immunological signatures have already been defined for successful vaccines, such as the yellow fever vaccine28. The hope is that signatures should be invaluable to guide the design of more effective vaccines and to evaluate or improve those vaccines already available. However, so far, predictive signatures have had limited success in guiding the development of improved or novel vaccines or explaining why existing vaccines fail in some individuals but not in others. This topic of systems vaccinology enjoys great enthusiasm and support, and exciting discoveries emerge almost daily. For example, as this review was being written, a report provided a molecular signature to explain why some influenza vaccine recipients develop clinical adverse responses29. With regard to the value of systems biology, the future is bright, but meaningful success remains a vexation.\n\n\nCan the microbiome explain almost everything?\n\nFew topics currently enjoy more journal space in immunology than the realization that microbes and other residents at mucosal and surface sites can influence systemic immune responses to many antigens that include viral infections and vaccines30. With viral infections, a report by Iwasaki showing that the microbiome of the gut impacted on immunity to influenza in the lung was the beginning31, but many reports along similar lines have followed. Of particular interest to viral immunology is that viruses can influence the outcome of infections to other types of agents. For example, herpesvirus latency can impact on the outcome of infection with Listeria bacteria and parasitic worms32, and norovirus infection influences the outcome of enterococcal infection33. An insightful and thought-provoking review on this topic, which is referred to as transkingdom metagenomics, was published recently by Pfeiffer and Virgin34. The translational application of the field is that manipulating the microbiome (and/or other “omes”) might improve the success of vaccines and perhaps other immunomodulators. However, as argued by Hanage35, the field requires a healthy dose of skepticism. This includes the need to decide between cause and mere correlation, the issue of whether experiments detect differences that matter and reflect reality, as well as the question of whether anything else could explain the findings. On balance, however, we remain optimistic that in the long term translational benefits to the viral immunology field will accrue. Indeed, it could be that bugs will be useful as therapies to manage certain viral infections.\n\n\nSome conclusions\n\nThe field of viral immunology is alive and well, and researchers continue to make significant and exciting contributions to our understanding of the fundamental biology of the immune system. Yet the practical translational application of this fascinating and fast-moving area of science is just a little disappointing. Some novel vaccine formulations have been developed, but far more are needed. We have also been successful in generating useful immunomodulators, but invariably their cost is prohibitive and beyond the reach of most persons. We have tooled up to respond briskly to new viral emergences such as Ebola and Zika, but in reality making and getting permission to test new vaccines and therapies remains a slow process. We also need to fully douse the idea held by some in society that the effective vaccines we use cause more harm than good. The introduction of “improved” vaccine formulations with fewer side effects may have the unwanted consequences of reduced efficacy. One example of this is the shift from whole-cell to acellular pertussis vaccines, which it now appears are not providing optimal control of pertussis. Moreover, loss of herd immunity within communities in developed countries owing to reduced vaccination rates, as parents and even doctors lose sight of the risks of not vaccinating, remains a clear and present danger. Nevertheless, the tools to develop and design new vaccines and predict efficacy continue to improve as our basic understanding of the immune system advances. As the promise and enthusiasm for new vaccines and drugs continue to drive research funding and public health agendas, the outlook suggests that there is much on the horizon.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nBarry T. Rouse is supported by grants from the National Institutes of Health and Scott N. Mueller by the Australian Research Council and the Australian National Health and Medical Research Council.\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\nGraham BS, Modjarrad K, McLellan JS: Novel antigens for RSV vaccines. Curr Opin Immunol. 2015; 35: 30–8. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRappuoli R, Bottomley MJ, D'Oro U, et al.: Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design. J Exp Med. 2016; 213(4): 469–81. 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PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDuPage M, Bluestone JA: Harnessing the plasticity of CD4+ T cells to treat immune-mediated disease. Nat Rev Immunol. 2016; 16(3): 149–63. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nOhkura N, Hamaguchi M, Morikawa H, et al.: T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development. Immunity. 2012; 37(5): 785–99. PubMed Abstract | Publisher Full Text\n\nKoenen HJ, Smeets RL, Vink PM, et al.: Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells. Blood. 2008; 112(6): 2340–52. PubMed Abstract | Publisher Full Text\n\nBerod L, Friedrich C, Nandan A, et al.: De novo fatty acid synthesis controls the fate between regulatory T and T helper 17 cells. Nat Med. 2014; 20(11): 1327–33. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nYin Y, Choi SC, Xu Z, et al.: Normalization of CD4+ T cell metabolism reverses lupus. Sci Transl Med. 2015; 7(274): 274ra18. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLi S, Nakaya HI, Kazmin DA, et al.: Systems biological approaches to measure and understand vaccine immunity in humans. Semin Immunol. 2013; 25(3): 209–18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQuerec TD, Akondy RS, Lee EK, et al.: Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans. Nat Immunol. 2009; 10(1): 116–25. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKidd BA: Decoding the immune response to successful influenza vaccination. Nat Immunol. 2016; 17(2): 113–4. PubMed Abstract | Publisher Full Text\n\nCaballero S, Pamer EG: Microbiota-mediated inflammation and antimicrobial defense in the intestine. Annu Rev Immunol. 2015; 33: 227–56. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nIchinohe T, Pang IK, Kumamoto Y, et al.: Microbiota regulates immune defense against respiratory tract influenza A virus infection. Proc Natl Acad Sci U S A. 2011; 108(13): 5354–9. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBarton ES, White DW, Cathelyn JS, et al.: Herpesvirus latency confers symbiotic protection from bacterial infection. Nature. 2007; 447(7142): 326–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAbt MC, Buffie CG, Sušac B, et al.: TLR-7 activation enhances IL-22-mediated colonization resistance against vancomycin-resistant enterococcus. Sci Transl Med. 2016; 8(327): 327ra25. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPfeiffer JK, Virgin HW: Viral immunity. Transkingdom control of viral infection and immunity in the mammalian intestine. Science. 2016; 351(6270): pii: aad5872. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHanage WP: Microbiology: Microbiome science needs a healthy dose of scepticism. Nature. 2014; 512(7514): 247–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "14049",
"date": "27 May 2016",
"name": "Ann Arvin",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14050",
"date": "27 May 2016",
"name": "Robert Hendricks",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1015
|
https://f1000research.com/articles/5-1014/v1
|
27 May 16
|
{
"type": "Research Article",
"title": "Early-childhood housing mobility and subsequent PTSD in adolescence: a Moving to Opportunity reanalysis",
"authors": [
"David C. Norris",
"Andrew Wilson",
"Andrew Wilson"
],
"abstract": "In a 2014 report on adolescent mental health outcomes in the Moving to Opportunity for Fair Housing Demonstration (MTO), Kessler et al. reported that, at 10- to 15-year follow-up, boys from households randomized to an experimental housing voucher intervention experienced 12-month prevalence of post-traumatic stress disorder (PTSD) at several times the rate of boys from control households. We reanalyze this finding here, bringing to light a PTSD outcome imputation procedure used in the original analysis, but not described in the study report. By bootstrapping with repeated draws from the frequentist sampling distribution of the imputation model used by Kessler et al., and by varying two pseudorandom number generator seeds that fed their analysis, we account for several purely statistical components of the uncertainty inherent in their imputation procedure. We also discuss other sources of uncertainty in this procedure that were not accessible to a formal reanalysis.",
"keywords": [
"mental health",
"housing mobility",
"adolescent",
"post traumatic stress disorder"
],
"content": "Introduction\n\nThe Moving to Opportunity for Fair Housing Demonstration (MTO) was a social experiment mandated by Congress, and conducted during the 1990’s. From 1994 to 1998, 4,604 households residing in distressed inner-city housing in five U.S. cities were randomized to three experimental groups. A control group received no housing voucher; a traditional voucher group received a standard ‘Section 8’ housing voucher; and a low-poverty voucher group received an experimental type of voucher usable only for housing located in a low-poverty area. Adults and children in the MTO households were surveyed in 2001 and 2011 to assess a variety of economic and mental health outcomes.\n\nA 2003 MTO Interim Evaluation1 (4–7 years after randomization) revealed an interesting interaction between gender and housing mobility, with respect to outcomes of delinquency and risky behavior: girls benefited, but boys did not. This finding, somewhat at odds with earlier research2, spurred subsequent explanatory efforts3–6.\n\nIn 2014, Kessler et al.7 published an analysis of the Final Youth Evaluation (10–15 years after randomization), reporting statistically significant and numerically substantial voucher effects on several psychiatric diagnoses in boys. Compared with controls, boys from low-poverty voucher households exhibited elevated 12-month prevalence of PTSD (6.2% vs 1.9%; OR, 3.4 [95% CI, 1.6–7.4]) and ‘conduct disorder’ (6.4% vs 2.1%; OR, 3.1 [95% CI, 1.7–5.8]). One of us (DCN) has previously suggested8 that these results are open to criticism on construct validity9 grounds. While working to define a protocol for empirically exploring this question, however, DCN learned that the PTSD outcome in 7 was imputed in a manner that invites scrutiny at the more basic level of statistical conclusion validity10.\n\nAlthough the report by Kessler et al.7 describes in some detail the imputation of missing covariates in the Final Youth Evaluation, it does not indicate that its PTSD outcome was in fact imputed. This imputation initially came to attention through a footnote on page 38 of 11, and was subsequently confirmed in a (Nov 2014) communication from the original authors.\n\nThe MTO Final Youth Survey employed an abridged, computerized self-administered version of the Composite International Diagnostic Interview (CIDI)12. The full version of the CIDI was used in the National Comorbidity Survey Adolescent Supplement (NCS-A), where its PTSD diagnostic algorithm was found to have a \"moderate\" concordance (AUC, 0.79) with diagnoses obtained through clinical diagnostic interviews13. The abridgment of the MTO instrument prevented direct application of the CIDI diagnostic algorithms in the MTO, however. Instead, CIDI algorithm-derived lifetime PTSD diagnoses in the NCS Replication survey (NCS-R) were regressed on responses to those PTSD-related questions which were retained in the MTO instrument, and the resulting (logistic) regression model was used to impute PTSD diagnoses for the MTO Final Youth Survey respondents. (In addition to the retained PTSD-related questions, the ‘obligatory’ variables age, sex and race were also included as regressors; see Table 1). Imputation was performed by Bernoulli draws consistent with the (logit) probabilities yielded by this regression equation. Responses to questions about recency of symptoms were in turn used to compute 12-month prevalence diagnoses from these imputed lifetime diagnoses.\n\nA number of objections to this outcome imputation procedure can be articulated without recourse to a reanalysis.\n\nThe imputation is superfluous. Viewed as a link in the chain of the analysis, the imputation itself appears superfluous. It introduces an information-destroying and noise-generating transformation of the predictive model’s real-valued outputs—logit probabilities on the continuous interval (–∞, ∞)—to pseudorandom ‘outcomes’ in the set {no, yes}. In order to interpret conclusions about these imputed outcomes as if they were conclusions about real PTSD, it would be necessary to defend the outputs of the predictive model as genuine probabilities. But if these were true probabilities, then they could be analyzed directly, without interposing a noisy and information-destroying pseudorandom number generation (RNG) step.\n\nIt makes the reported CIs strictly artifactual. Because a logistic regression model has no error term, the confidence intervals reported for the logistic regression of imputed PTSD on voucher treatment in Kessler et al.7 convey only the uncertainty arising from the RNG sampling performed to impute the PTSD outcomes. All of the substantive sources of uncertainty lurk in the specification and estimation of the imputation model, procedures that are opaque to a reader of 7.\n\nThe model specification appears desultory. The specification of this outcome-imputation model gives every impression that it was regarded as purely phenomenological, and that it was specified, estimated and checked in the desultory manner customary for imputation of missing covariates. The model shows no evidence of an attempt to include additional predictors (beyond the ‘obligatory’ age/sex/race) that the MTO Final Youth Survey shared with NCS-R. No bootstrap-validation is described, nor is any other investigation of the predictive performance of the model in the NCS-R population against which it was estimated. No shrinkage was applied to correct for overfitting14. The model coefficients themselves (see Table 1) seem uninterpretable in causal terms, unless it can be supposed (e.g.) that being badly beaten in childhood confers protection from PTSD in nearly equal measure to the PTSD risk incurred from a sexual assault/molestation.\n\nGeneralizability of NCS-R to MTO is doubtful. Even if the use of an outcomes-imputation model had been described in 7, and if its performance had been rigorously investigated and overfitting addressed, the question of generalizability from NCS-R to MTO populations would still remain open. Most plainly, the NCS-R and MTO Youth populations barely overlap in age (Figure 1). Also important may be the qualitative differences expected between the general-population sample of NCS-R and the inner-city MTO Youth sample, with regard to their traumatic exposures and their sources of resilience relevant to PTSD.\n\nThe PTSD imputation model used in 7 was estimated in the former population, and applied to the latter.\n\n\nMethods\n\nTo develop a focused critique in an objective form, we undertook a reproduction and reanalysis of the PTSD findings in 7.\n\nIn an August 2015 communication to the original authors, we committed to principles of reanalysis articulated by Christakis and Zimmerman15. Of greatest importance, to avoid a “statistical fishing expedition”15, we committed to limiting the scope and methods of our reanalysis to those discussed in an earlier communication (Nov 2014), which is basically recapitulated in points 1–4 above. Our commitment incorporated an explicit exclusion of multiple aspects of the original analysis: “We do not intend to critique your multiple imputation of partially missing covariates, nor the weighting techniques you employed, nor your case-level imputation to correct for non-response. We will take these as givens, as manifested in your SAS code and in the precomputed weights in the data” (Aug 2015). Additionally, regarding as too weak Christakis and Zimmerman’s requirement that original authors “should be provided with the opportunity to review and comment on the reanalysis before its acceptance for publication”15, we committed in this same communication also to giving the original authors “access to all of our reanalysis code no later than our manuscript is submitted for peer review.” (Said access was provided on May 3, 2016.) Finally, to avoid publication bias, authors committed to “make all reasonable efforts to publish a manuscript describing our reanalysis findings regardless of the ‘significance level’ of the widened confidence intervals it ultimately yields.”\n\nOur analytic code and results were maintained in a repository on GitHub, with the primary intent to support reproduction and scrutiny of our reanalysis by the original authors, as well as by reviewers and other third parties. All statistical code provided by the original researchers was checked-in to this repository exactly as received, so that any subsequent modification made by us could be readily inspected via the git diff command. A README file in the root directory of this repository provides orientation to directory structure and repository content. A single SAS script was developed which reproduces all steps of our reanalysis.\n\nWe resampled the coefficients of the PTSD imputation model from a multivariate normal distribution with mean and covariance matrix as provided by the original authors in a Jan 2016 communication. The first ‘sample’ was replaced by the coefficients in Table 1, so as to embed a formal reproduction of the original effect estimates within the larger bootstrapping exercise.\n\nWe varied the seed used in generating the pseudorandom U(0, 1) probability thresholds necessary for imputing a binary PTSD ‘outcome’ from the (logit) probabilities produced by the PTSD imputation model. In 7, this seed was set to 1234567; we allowed this ‘pr_seed’ to range over {123, 1234, 12345, 123456, 1234567}.\n\nWe varied the seed used for multiple imputation of missing covariates. In 7, the seed value was 524232; we allowed this ‘mi_seed’ to range over the 10 consecutive values 524230–524239.\n\nTo explore also the impact of arbitrary PTSD model specification, we performed a similar bootstrapping exercise where, instead of resampling the model coefficients, we explored a small set of alternate specifications of the imputation model. A 2×2×2 grid of models was explored in which: (1) age was or was not included; (2) race was or was not included; and (3) the NCS-R sample was or was not restricted to age ≤ 40 before estimating the model coefficients. The eight models thus produced were designated as in Table 2; model specification a1r1s99 accords with that used in 7.\n\nModel ‘a1r1s99’ is the original specification.\n\nBecause the standard MTO data package maintained by the Department of Housing and Urban Development (HUD) contains pre-imputed data, whereas the original analysis in 7 employed multiple imputation of the missing values in raw data, a specially prepared MTO data package was required for this work. The National Bureau of Economic Research (NBER) kindly prepared and archived this package with HUD.\n\n\nResults\n\nWe reproduced the originally reported odds ratios and 95% confidence intervals for voucher effects on 12-month prevalence of PTSD in boys: 3.44025 [1.60147–7.39026] and 2.67817 [1.23268–5.81873] for the low-poverty and traditional vouchers, respectively. (These figures were reported to 1 decimal place in the original article.)\n\nIn the course of achieving this reproduction, several unexpected features of the original analysis came to light: (1) the imputation of PTSD outcomes preceded the 20× multiple imputation of missing covariates; (2) 24.5% (456/1863) of the MTO boys were uninterviewed, and so contributed little more than their baseline characteristics to the analysis.\n\nFigure 2 shows our 10 × 5 × 10 = 500 bootstrapped estimates of the low-poverty voucher effect on 12-month PTSD prevalence in MTO boys, placing the original estimates (shown in red) in context.\n\nFigure 3 shows our 8 × 5 × 10 = 400 estimates of the low-poverty voucher effect on 12-month PTSD prevalence in MTO boys, bootstrapped over alternative specifications of the PTSD imputation model.\n\nEach panel contains 10 effect estimates obtained by resampling the PTSD imputation model as described in the text, with the first ‘resample’ being the original coefficients as in Table 1. For each panel, the green strip shows the RNG seed used to generate pseudorandom U(0, 1) thresholds for PTSD imputation, and the orange strip shows the RNG seed used for the multiple imputation of missing covariates. In 7, these seeds were set to 1234567 and 524232, respectively; thus, the effect drawn in red represents our reproduction of the published effect estimate. Note the logarithmic scale.\n\nEach panel contains eight effect estimates obtained using the alternative PTSD imputation model specifications in Table 2. For each panel, the green strip shows the RNG seed used to generate pseudorandom U(0, 1) thresholds for PTSD imputation, and the orange strip shows the RNG seed used for the multiple imputation of missing covariates. In 7, these seeds were set to 1234567 and 524232, respectively; thus, the effect drawn in red represents our reproduction of the published effect estimate. Note the logarithmic scale.\n\nEach iteration of our bootstrap required just over 4 minutes on a modern Windows workstation; Figure 2 and Figure 3 thus represent about 35 and 28 hours of computation, respectively.\n\nA public fork of our code repository may be accessed at https://github.com/DNC-LLC/MTOpublic.\n\n\nDiscussion\n\nA social science research enterprise as massive as the MTO Demonstration necessarily involves manifold layers of analysis that may obscure the provenance of analytical results and of associated scientific ‘findings’. This situation creates a compelling rationale for reproduction and reanalysis of such research. The work presented here is, we believe, the first independent attempt to reproduce or reanalyze published results from MTO. (HUD informed us that our March 5, 2015 application for an MTO data license was the first such request received by them.)\n\nA frequentist confidence interval should be understood as abstracting away the arbitrariness immanent in a point estimate, by situating that estimate within the context of an ensemble of imagined ‘possible replications’. Regrettably, in much current statistical practice only the arbitrariness of a sampling procedure typically is recognized and objectively accounted for 16. Yet other forms of arbitrariness, such as overfitting and model selection, are equally amenable to similar ‘contextualization’ by bootstrapping techniques such as we employ here. (Indeed, we have structured our reanalysis so as to accentuate this frequentist abstracting-and-contextualizing idiom; our Figure 2 and Figure 3 present a direct visual analogue of the ensemble, which we hope will prove broadly accessible to social scientists. A reanalysis performed strictly for a professional statistical audience would dispense with explicit seed variation, instead simply increasing the number of multiple imputations performed from 20 to 1000; it would likewise have done away entirely with the imputation of a binary PTSD outcome, substituting a direct analysis of the PTSD probabilities.)\n\nThe confidence intervals reported in 7 account only for the sampling inherent in the MTO design. Our bootstrapping analysis further abstracts away (1) arbitrariness arising when Kessler et al. fixed their PTSD imputation model at its maximum-likelihood estimate as if known with infinite precision; (2) the arbitrariness of what appears to have been a desultory model specification; (3) some purely technical forms of arbitrariness appearing in the form of sensitivity to RNG seed selection. While we have achieved only partial abstraction of these forms of arbitrariness, and have not addressed overfitting, it does appear that the original claim of ‘statistical significance’ for a voucher-on-PTSD effect in MTO boys withstands the formal challenge of our reanalysis as planned. That is to say, a randomly selected confidence interval from Figure 2 or Figure 3 typically sits above the OR = 1 threshold.\n\nWhat is clear enough, however, is that a statistical analysis that randomly selects its ‘findings’ from Figure 2 or Figure 3 cannot be recommended for its wholesome frequentist properties. Indeed, the evident clustering of variance within certain (combinations of) seed choices disallows any straightforward attempt to compute a single, wider confidence interval from these bootstrap samples.\n\nThe main contribution of our reanalysis may be simply to bring enough transparency to an otherwise obscure PTSD imputation procedure (1) to avert its application in further research, and (2) to encourage exploration of alternative modes of analysis of the interesting and important social-scientific and policy questions surrounding the MTO boys’ exposures—and responses—to violence.\n\nThe close scrutiny involved in a reanalysis of this kind inevitably brings to light aspects of the original analysis which could not have been anticipated, provoking questions not pre-specified as part of the reanalysis. We are now interested to know the statistical impact of including in the original analysis7 (and likewise in the present reanalysis) 456 uninterviewed MTO boys for whom little more than baseline covariates were available—especially in light of the fact that the PTSD imputation preceded the multiple imputation of missing covariates. We hope to address this question in a subsequent analysis.\n\n\nConclusions\n\nWe have reproduced and reanalyzed a key finding reported by Kessler et al.7, following sound principles of reanalysis15 with particular attention to limiting our scope and methodology to issues and techniques proposed before we examined the data. The original claim of a statistically significant effect of the low-poverty voucher on PTSD in adolescent boys has formally withstood the objective challenge presented by this reanalysis. Our reanalysis also brings new transparency to outcomes-imputation methods employed in 7, which may contribute usefully to the evaluation of this work by researchers and policy analysts.\n\n\nData availability\n\nOpen Science Framework: Dataset: Early-childhood housing mobility and subsequent PTSD in adolescence: a Moving to Opportunity reanalysis, doi 10.17605/osf.io/jcpyn17",
"appendix": "Author contributions\n\n\n\nDCN developed the concept and design of the reanalysis, supervised the study, and drafted the manuscript. AW revised the manuscript for important intellectual content and supervised the study’s secure computing environment. Both authors contributed equally to the statistical programming, data analysis and interpretation.\n\n\nCompeting interests\n\n\n\nThe authors have no financial, personal, or professional competing interests to declare in connection with this article.\n\n\nGrant information\n\nDCN received partial funding for travel to Salt Lake City from the University of Utah, through AW’s Faculty Development Grant.\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 wish to acknowledge Nicole Bailey of Anolinx, for her assistance with data licensing and acquisition, and for drawing our attention to the difference in age distributions illustrated in Figure 1; Dr. Ronald C. Kessler and his team for their kind cooperation with this reanalysis, very special thanks being due to Nancy A. Sampson for her many timely and helpful communications with us particularly regarding gaps in the public versions of NCS-R and MTO data; Matt Sciandra of NBER for preparing the special package of data and SAS code which proved essential for our reproduction and reanalysis; Anolinx for providing the secure workstation on which this work was done.\n\n\nReferences\n\nOrr L, Feins JD, Jacob R, et al.: Moving to Opportunity for Fair Housing Demonstration Program: Interim Impacts Evaluation. Technical report, U.S. Department of Housing & Urban Development, Office of Policy Development & Research, Washington, DC, 2003. Reference Source\n\nKatz LF, Kling JR, Liebman JB: Moving to Opportunity in Boston: Early Results of a Randomized Mobility Experiment. Q J Econ. 2001; 116(2): 607–654. Publisher Full Text\n\nKling JR, Liebman JB, Katz LF: Experimental Analysis of Neighborhood Effects. Econometrica. 2007; 75(1): 83–119. Publisher Full Text\n\nPopkin SJ, Leventhal T, Weissman G: Girls in the ’Hood: The Importance of Feeling Safe. Brief 1, The Urban Institute, Washington, DC, 2008. Reference Source\n\nJackson L, Langille L, Lyons R, et al.: Does moving from a high-poverty to lower-poverty neighborhood improve mental health? A realist review of ‘Moving to Opportunity’. Health Place. 2009; 15(4): 961–970. PubMed Abstract | Publisher Full Text\n\nClampet-Lundquist S, Kling JR, Edin K, et al.: Moving teenagers out of high-risk neighborhoods: how girls fare better than boys. Am J Sociol. 2011; 116(4): 1154–1189. PubMed Abstract | Publisher Full Text\n\nKessler RC, Duncan GJ, Gennetian LA, et al.: Associations of housing mobility interventions for children in high-poverty neighborhoods with subsequent mental disorders during adolescence. JAMA. 2014; 311(9): 937–948. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNorris DC: Housing mobility and adolescent mental health. JAMA. 2014; 312(2): 190. PubMed Abstract | Publisher Full Text\n\nCronbach LJ, Meehl PE: Construct validity in psychological tests. Psychol Bull. 1955; 52(4): 281–302. PubMed Abstract | Publisher Full Text\n\nShadish WR, Cook TD, Campbell DT: Experimental and quasi-experimental designs for generalized causal inference. Houghton Mifflin, Boston, 2001. Reference Source\n\nMeasuring DSM-IV mental disorders in the Moving To Opportunity Final Evaluation 11/26/2013. Accessed 4/21/2016. Reference Source\n\nKessler RC, Ustün TB: The World Mental Health (WMH) Survey Initiative Version of the World Health Organization (WHO) Composite International Diagnostic Interview (CIDI). Int J Methods Psychiatr Res. 2004; 13(2): 93–121. Publisher Full Text\n\nKessler RC, Avenevoli S, Green J, et al.: National comorbidity survey replication adolescent supplement (NCS-A): III. Concordance of DSM-IV/CIDI diagnoses with clinical reassessments. J Am Acad Child Adolesc Psychiatry. 2009; 48(4): 386–399. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSteyerberg EW: Clinical Prediction Models. Statistics for Biology and Health. Springer New York, New York, NY, 2009. Publisher Full Text\n\nChristakis DA, Zimmerman FJ: Rethinking reanalysis. JAMA. 2013; 310(23): 2499–2500. PubMed Abstract | Publisher Full Text\n\nGreenland S: Multiple-bias modelling for analysis of observational data. J R Stat Soc A. 2005; 168(2): 267–306. Publisher Full Text\n\nNorris D, Wilson A: Dataset: Early-childhood housing mobility and subsequent PTSD in adolescence: a Moving to Opportunity reanalysis. Open Science Framework. 2016. Data Source"
}
|
[
{
"id": "14046",
"date": "24 Jun 2016",
"name": "Ewout W. Steyerberg",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 timely paper in this era of debate on scientific integrity and transparency. The paper is a good illustration of what can be achieved by data sharing and rigorous re-analysis.\n\nSpecifically, the issues of dealing with missing data are relevant, and especially outcomes-imputation methods. Imputation of missing outcome is anyway controversial; Von Hippel proposed a Multiple Imputation then Deletion (MID) approach, which has some appeal but was not supported by a recent study, which argued that auxiliary variables could be beneficial in this situation to impute missing Y (Sullivan, 2015). The Von Hippel and Sullivan papers need to be discussed briefly as educational efforts in the current paper, in line with the paragraph: The imputation is superfluous.\n\nThere is 2 main issues in the specific hypothesis studied related to housing vouchers: effect estimate and uncertainty in the effect.\nThe MTO project had a strong design, with randomization to 3 groups for evaluation of the intervention. The specific outcomes highlighted here are psychiatric diagnoses, among which PTSD; this was one of many outcomes evaluated perhaps, and not the primary outcome of this study. This brings in a multiple comparisons problem, which may be emphasized more by the current authors.\nWe now focus on one association that stood out among many associations that were probably evaluated. First we may wonder whether we should adjust formally for multiple testing, such as a classical Bonferroni correction for the p-value. Second, we know that the strength of the association will be overestimated, even if a true association.\nThe suggested boys vs girls interaction further complicates the multiple comparison issue; essentially all analyses are doubled. I would like some further discussion on these broader issues of interpreting research findings (Ioannadis, 2014).\n\nUncertainty is usually expressed as a 95% confidence interval; the current study appropriately highlights that multiple sources of uncertainty may need to be considered, specifically in the context of multiple imputation.\nOverall, this paper is an interesting contribution to statistical aspects on modern analysis. It illustrates that different researchers may use different methods and approaches. It also shows some progress in scientific methods, e.g. the use of bootstrapping is now more feasible than some years ago due to ever increasing computational power. This type of reanalysis is to be applauded and stimulated to increase the credibility of scientific research findings.",
"responses": []
},
{
"id": "14561",
"date": "15 Jul 2016",
"name": "Fred Hasselman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 discusses a re-analysis and subsequent re-interpretation of a previously published study on the important topic of PTSD in adolescence. The authors argue the imputation method used in the original article is flawed and I agree with their objections.\nThe scientific method is supposed to to be self-correcting; the lucid problem analysis and subsequent solutions presented in this article and decision to disseminate it through a platform like F1000Research are examples of \"open science\" practices that will enable us to achieve this goal for our discipline.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1014
|
https://f1000research.com/articles/5-1013/v1
|
27 May 16
|
{
"type": "Research Article",
"title": "Effects of flanker type and position on foveal letter recognition",
"authors": [
"Mohd Izzuddin Hairol",
"Qazi Mohamad Omair",
"Sharanjeet Kaur",
"Qazi Mohamad Omair",
"Sharanjeet Kaur"
],
"abstract": "Our ability to identify a foveally viewed letter at resolution threshold reduces when flankers are placed at a certain distance from it, compared to when it is presented on its own. In this study, we investigated how type and position of flankers influences foveal letter recognition. We measured participants’ performance to identify unflanked Sheridan-Gardiner letters (A, H, O, U, T, V or X), using a seven-alternative-forced-choice paradigm with the method of constant stimuli to obtain 80-90% correct response. Performance was measured again to identify a target letter in the presence of different flanker types. Flankers were bars and letters (placed either to the left and right of the target, above and below the target or on four sides of the target), and a surrounding box. Separation between the target letter and flankers varied between zero (abutting) and ten stroke widths (two letter sizes). For all flanker types, separation between the target ad the flankers significantly influenced performance (all p < 0.0001). Flankers abutting the target and at separation of one stroke width caused the largest reduction in performance, regardless of flanker type. For bar flankers, the largest drop in performance (up to 50 percent) occurred with bars placed on all four sides of the target. For letter flankers, flankers placed above and below the target reduced performance as much as four surrounding flankers. At separation of one stroke width, flanking letters and bars caused a further 10% reduction in performance than a surrounding box. Our results would have significant implications on the design of crowded visual acuity charts, especially those that are available on the market, which have different crowding features between them.",
"keywords": [
"Letter recognition",
"contour interaction",
"crowding",
"visual acuity"
],
"content": "Introduction\n\nClinically, crowding is the reduction in visual acuity for a target letter presented together with other crowding features, such as other letters, a surrounding box or contours, compared with acuity measured with single letters (Flom et al., 1963; Stuart & Burian, 1962). Flom et al. (1963) reported that performance for localising the gap of a Landolt C, at acuity level, is reduced by flanking bars that are placed less than one optotype width away from the Landolt C. This effect has been specifically named as contour interaction, to describe the effects of surrounding contours on acuity. Crowding and contour interaction are sometimes used interchangeably, although the latter is considered as a subset of crowding and is usually defined specifically as reduction in acuity by nearby contours (Danilova & Bondarko, 2007; Flom et al., 1963; Flom, 1991; Formankiewicz & Waugh, 2013). Besides contour interaction, other factors that influence crowding effects include gaze control inaccuracy and attentional factors (Flom et al., 1963).\n\nWhen tested with commercially available paediatric acuity tests, Norgett & Siderov (2014) reported that contour interaction had a greater effect in children than in adults. The younger children group (up to 6 years 9 months in age) had worse acuity for crowded letter charts compared to the older children group (up to 9 years 8 months in age), which could be explained by their underlying development of accurate gaze control and fixational eye movements. Other studies have also reported that contour interaction occurs over a larger distance in children than in adults (Jeon et al., 2010; Semenov et al., 2000). This suggests contour interaction (and crowding), and their associated factors including accuracy of fixational eye movement and attention are attributable to age.\n\nTypically, the crowding effects described above were measured with the flanking features surrounding all four sides of a target letter (or letters). However, it has been shown that in a square C acuity task, performance of adult observers was differently affected by two flanking bars, dependent on whether they were placed orthogonal or parallel to the gap of the C (Liu, 2001). This indicates that although the oculomotor and attentional components have matured in adult observers, contour interaction effects are still dependent on the relative position of the crowding features from the target.\n\nWhen measured in the peripheral vision of adults, crowding effects are greater in magnitude when the flankers are similar to the target letter, compared to when they are dissimilar (Bernard & Chung, 2011; Kooi et al., 1994; Leat et al., 1999). In the adult fovea, however, conflicting results on the effects of flanker type on crowding magnitude have been reported. In the adult fovea, crowding effects can be similar in magnitude, regardless of whether the flankers that are different or similar to the target letter (Leat et al., 1999; Norgett & Siderov, 2014). Norgett & Siderov (2011) showed that, in adults, crowding effects are similar for single letters surrounded by either four bars or four letter flankers, placed half a letter width away. However, it is unclear if crowding is still similar when the number of flankers is reduced and when they are independently placed at various distances from the target letter. For close target-flanker separations, letter flankers can result in greater levels of crowding (e.g. Lalor et al., 2016) which could be due to greater similarity between the flankers and the target (Bernard & Chung, 2011; Kooi et al., 1994).\n\nKnowledge on crowding and contour interaction has been applied in the design of crowded acuity tests, based primarily on the results reported by Flom et al. (1963). Flom et al. reported that the largest reduction in performance to discriminate the gap of a Landolt C occurred when contours were placed at edge-to-edge distance of two stroke widths away from the target. In paediatric patients, accurate measurement of visual acuity is important to detect visual conditions such as amblyopia, hence allowing accurate monitoring of the response to any prescribed treatment. Amblyopia is clinically diagnosed using acuity tests designed to induce crowding, as it is known that crowding effect may be greater in amblyopes than in patients with normal vision (Hess et al., 2001; Levi et al., 2002). Commercially available crowded acuity tests however, used different target-flanker spacing, flanker type, and optotype arrangement. Generally, in children’s acuity charts, flankers (which could be a surrounding box, other letters or contour) are placed at half an optotype width away or one optotype width away from the target. The target itself could be a letter, a string of letters, pictures or symbols and the surrounding flankers could be other letters, a box, or the combination of both (Atkinson et al., 1988; Kay, 1983; Salt et al., 2007). These differences may produce different amount of crowding exerted possibly affecting their sensitivity to detect amblyopia.\n\nThe aims of this study were (1) to compare crowding produced by different number of flankers and by different flanker types, and (2) to determine the optimum separation between target and flankers that produce maximum crowding. The results would have an application in designing a clinical letter test that would exert maximum crowding effects. The test could then be more sensitive in detecting visual conditions that are characterised by crowding, such as in amblyopia, where acuity is worse when tested with a crowded target than when it is measured with isolated letters.\n\n\nMethods\n\nEight participants were involved in this study, based on convenience sampling. All participants had best-corrected visual acuity of 6/6 or better in both eyes, healthy, and had no significant ocular or systemic diseases. This research was approved by Universiti Kebangsaan Malaysia’s Researc Ethics Committee (NN-2014-083), which complied with the tenets of the Declaration of Helsinki. All participants provided written informed consent before the start of the experiment.\n\nThe experiment was run on a Macintosh desktop, where Matlab (version R2012a) with PsychToolbox version 3.0.8 extension (Pelli, 1997) was installed to create the experimental stimuli. Stimuli were digitised Sheridan Gardiner letters (A, H, O, T, U, V, and X), scanned from their printed clinical chart format. Each letter had a 5×5 construction, that is, each stroke was one fifth of the letter size and each letter had equal width and height. The Weber’s contrast of the letters was 90%. Stimuli were displayed on a Samsung CRT monitor running at 100 Hz.\n\nIn this study, we measured contour interaction and crowding functions for visual acuity (Flom et al., 1963). First, we measured performance of each participant to identify an isolated target letter. The physical size of the letter displayed on the computer screen was fixed. Isolated letters were presented randomly for 100 trials and participant’s responses were recorded. A viewing distance was chosen (between 11 and 13 metres) so that the percent correct response fell between 80 to 90 percent for each participant. All participants completed four 100-trial runs. Performance across the four runs was averaged to obtain the unflanked (i.e. uncrowded) performance.\n\nNext, the participant’s task was to identify the target letter in the presence of crowding features. When the target letter was flanked by bars, the separation between them was expressed in edge-to-edge separations of 0, 1, 2.5, 3.5, 5, and 10 stroke-widths (each stroke-width is equivalent to one fifth of the target letter size). The bars were placed either horizontally, that is, to the left and right of the target letter (Figure 1a); vertically, that is, above and below the target letter (Figure 1b); or at the top, bottom, left and right to the target letter (Figure 1c).\n\na. Target letter H flanked horizontally by two vertical bars. The size of each stroke of the letter (x) is one fifth of the whole letter size (which is 5x). The width of the flanking bars is one stroke-width and the length of the bars is the same to that of the target letter. The separation shown here is one stroke-width, where separation is measured from the closest edge of the target letter to the closest edge of the flanking bars. b. Target letter T flanked vertically by two horizontal bars. c. Target letter A flanked by four bars on all its sides. d. Target letter V flanked horizontally by two other letters. The centre-to-centre separation between target and flankers shown here is one letter width, which is equivalent to edge-to-edge separation of zero stroke-width, i.e. the target abuts the flankers. e. Target letter X flanked vertically by two other letters. f. Target letter U flanked on four sides by four other letters. g. Target letter O enclosed in a crowding box. The stroke-width of the box is the same as the stroke-width of the target letter. The separation between the closest edge of the letter and the box shown here is one stroke-width.\n\nFor letter flankers, the separation between the target letter and its flankers were calculated as the distance from the centre of the target to the centre of one of the flanking letters. Hence, separations between them were expressed in centre-to-centre units, which were 1, 1.25, 1.5, 1.75, 2 and 3. Note that centre-to-centre separation of 1 is equivalent to zero stroke-width in edge-to-edge units (i.e., two closest edges of the target and flanking letters were abutting). The flanking letters were placed either horizontal to the target letter (to the left and right of the target letter, Figure 1d); vertical to the target letter (above and below it, Figure 1e); or, at the top, bottom, left and right of the target letter (Figure 1f). Additionally, performance was also measured when the target letter was enclosed in a box (Figure 1g). The stroke of the box was one-fifth of the target letter size. The crowding features were always of the same contrast as the target letter. When letters were used as the crowding feature, they were never the same letter as the target and were always different from each other.\n\nTable 1 summarises the separation between target and flankers used in the study, expressed in edge-to-edge units for bar flankers and surrounding box, and centre-to-centre units for letter flankers. Conversion from centre-to-centre units to edge-to-edge units is also shown, to allow direct comparisons of the effects of the different types of flankers on letter recognition.\n\nAll participants completed four runs of each crowding condition. There were 100 trials in each run. Within each run, the target letter and the separation between the target and its crowding feature was randomised. In this part of the experiment, isolated letters (i.e. unflanked) were also included in some of the trials, with an equal probability of being displayed as the flanked letter trials. All experimental runs were conducted in a systematic order. Runs with flanking bars were completed first, followed by runs with flanking letters and runs with the surrounding box. In total, each participant completed 3200 trials, excluding practice trials.\n\nParticipants indicated their response verbally. The response was then entered by the examiner using a keyboard. No feedback was given. Viewing time was unlimited, that is, participants were allowed to take their time to identify the target letter, although generally the responses were prompt. With each keyboard input, the stimulus display was replaced by a mean luminance screen that appeared for 500ms before the presentation of the next trial. Testing was done monocularly using the subject’s dominant eye, determined with the hole-in-hand test (Miles, 1930). The non-viewing eye was covered with a black occluder. All participants were given training with the task and data collection only commenced after they were comfortable with the experimental procedures (at least two hours of training). Data collected during the training period were not included in the final analysis.\n\nRaw data were sorted in MS Excel version 14 and plotted in IgorProTM version 6.3.6.4 (WaveMetrics, Portland, OR). Statistical analyses were carried out using IBM Statistical Package for Social Sciences (SPSS) version 19.0. Data were analysed using repeated measures Analysis of Variance (ANOVA) with Greenhouse-Geisser correction to compare change in performance obtained with the different flanker types and positions. Post hoc pairwise comparisons with Benferroni corrections were performed where appropriate. Alpha level of 0.05 was used for all statistical tests.\n\nFigure 2 shows relative performance to identify the target letter as a function of target-flanking bar separation. Relative performance was calculated by subtracting the performance with flanking bars from performance to recognise an unflanked target. Therefore, performance lower than 0% correct for a flanked condition indicates a performance lower than that found for unflanked condition, i.e. crowding. Likewise, performance greater than 0% correct for a flanked condition indicates that participants’ correct responses for a flanked condition were higher than an unflanked condition.\n\nRelative performance was also measured for a target flanked by four bars (open circles). Performances are shown as a function of target-flanker separation.\n\nRepeated measures ANOVA revealed significant main effects of position [F(1.67, 11.71) = 9.43, p = 0.005] and target-flanker separation [F(1.74, 12.17) = 21.05, p < 0.001) on relative performance. The interaction between the position of flanking bars and target-bar separation was significant [F(3.58, 25.07] = 3.95, p = 0.02]. That is, the change in relative performance for identifying the target across separation was influenced by the position (and number) of the flanking bars. Two flanking bars placed at the bottom and top of the target (open triangles in Figure 2) exerted the least crowding effect that was significantly different from that obtained with four flanking bars [F(1,7) = 13.07, p < 0.01] . Performances with two flanking bars placed to the left and right of the target (open diamonds in Figure 2) and four bars surrounding the target (open circles in Figure 2) were not significantly different [F(1,7) = 5.14, p = 0.06]. The extent of crowding was also different dependent on the position of the flankers. Post hoc tests revealed that performance with top and bottom bars was significantly lower from unflanked conditions at separations of 0 (p=0.005) and 1 (p=0.001). With left and right bars, performance was significantly lower from unflanked conditions at separations of 0 (p<0.001), 1 (p=0.003) and 2.5 (p=0.006) stroke widths. With four surrounding bars, performance was also significantly lower from unflanked conditions at separations of 0 (p<0.001), 1 (p<0.001) and 2.5 (p=0.004) stroke widths.\n\nFigure 3 shows relative performance to identify the target letter as a function of separation between the target and flanking letters. Repeated measures ANOVA revealed that there was significant main effects of flanker position [F(1.36, 9.54) = 10.53, p = 0.006] and separation F(1.98, 13.88) = 62.54, p < 0.001] on relative performance. There was a significant interaction between flanking letter position and target-flanker separation [F(2.79, 19.56) = 6.54, p = 0.004]. Unlike with flanking bars, letter flankers placed at the top and bottom of the target letter (open triangles, Figure 3) exerted as much crowding as, and not significantly different from, that obtained with four letter flankers (filled circles in Figure 3) placed around the target [F(1,7) = 0.02, p = 0.88]. Least reduction of performance was obtained by two letter flankers that are placed to the left and right of the target (open diamonds in Figure 3) and it was significantly different from that obtained with four letter flankers [F(1,7) = 47.41, p < 0.001]. Post hoc tests showed that performance with left and right letter flankers was significantly lower than unflanked performance at separations of 0 and 1.25 centre-to-centre. For top and bottom, and four surrounding letter flankers, performance was significantly lower from unflanked conditions at separations of 0, 1.25 and 2.5 centre-to-centre (all p < 0.05). That is, the extent of crowding was larger for these two stimulus configurations.\n\nFigure 4 compares relative performances in identifying the target when it was flanked by four bars, four flanking letters and when it was surrounded by a box. For all types of flankers, the largest reduction in performance occurred when the flankers abutted the target. Generally, participants’ relative performance to identify the target letter was similar for a target flanked by either four letters or four bars. At separation of one stroke width, the surrounding box appeared to exert the least crowding effect compared to other types of flankers. Repeated measures ANOVA reveals that the interaction between flanker type and separation is significant [F(3.42, 23.92) = 3.15, p = 0.038), that is, the change in relative performance across separation was dependent on the flanking features used. Post hoc analyses revealed that a significant difference in performance with the different flanker types only occurred between letter flankers and box, when they abutted the target letter (p = 0.007), but not for bar flankers and at all other separations. The difference between flanked and unflanked performances occurred at a separation of 0, 1 and 2.5 stroke widths. This extent of crowding was similar to that found when four bars and four letter flankers were used.\n\nOur results are consistent with earlier findings that flanking bars reduced the recognition of a foveally fixated acuity target when they are in close proximity to each other (e.g. Flom et al., 1963; Stuart & Burian, 1962). Regardless of the type of the flankers used recognition of a target letter was similarly affected when there were four flankers surrounding it. Our results obtained with four surrounding letters and bars are consistent with the observations of Norgett & Siderov (2014) in adult observers. Other studies have also reported that with adult foveal viewing, crowding effects were similar for flankers that are categorically similar and dissimilar to the target (Danilova & Bondarko, 2007; Leat et al., 1999; Song et al., 2014). Norgett & Siderov (2014) reported that in children however, at separation half a letter width away (i.e. 2.5 stroke-widths) significantly larger foveal crowding effects were observed with letter flankers compared to four flanking bars. In our study, other than in the abutting condition, the largest reduction of performance occurred with target-flanker separation of one stroke-width. It is likely that larger crowding effects in children than adults would also be observed with flankers placed at one stroke-width away from the target.\n\nWe also showed that the reduction in correct performance was dependent on the number and relative position of the flankers surrounding the target. Other than the abutting condition, separation of about one stroke-width, edge-to-edge, exerts the most crowding effect for all conditions. However, at this target-flanker separation, the position of the flankers relative to the target letter influenced the magnitude of the crowding effect. Liu (2001) reported different patterns of performance for a square C orientation discrimination task when it was flanked by two bars that was dependent on the flanker’s position relative to the gap of the C, i.e. either parallel or orthogonal to the C’s gap. Therefore, if a crowded chart is to be developed using bars as the crowding feature, maximum crowding can be exerted with flanking bars placed either to the left and right or on all four sides of the target letter with a separation of one stroke width.\n\nWith two letter flankers, reduction in performance is significantly dependent on their position relative to the target letter. We observed the least crowding when the letters were place to the left and right of the target letter, especially when they were abutting and at approximately one stroke-width away from the target (Figure 3), compared to when they were placed above and below the target letter. Asymmetric horizontal-vertical crowding region have been reported when target and distractors are presented in the peripheral visual field (Feng et al., 2007; Hairol et al., 2014; Toet & Levi, 1992). An interesting observation is that two letter flankers placed at the top and bottom of the target letter reduced the participants’ performance just as much as having four letter flankers surrounding the target. Cherici et al. (2012) reported that for normal adults, prolonged fixation was inaccurate during foveal fixation, and the eye movement range when one was fixating may be larger than the spacing between letters at size threshold. It could be assumed that crowding would be similar regardless of whether a target letter is flanked vertically or horizontally by other letters. Indeed, a similar amount of crowding has been reported for a single letter surrounded by four letters on all its sides and for a string of five letters surrounded by other letters (Norgett & Siderov, 2014). Bedell et al. (2015) reported that more errors were made by adult observers when identifying a long string of letters compared to a short string of letters (all were arranged horizontally). However, it is unclear how error rate would differ for a string of vertically arranged letters. We showed that performance was reduced further for a target letter flanked vertically by other letters than that for letters that are flanked horizontally (Figure 3). As most adults are presumably more trained for reading letters that are arranged horizontally (as was the case for all participants in this study) there could be less fixation inaccuracies when letters are read horizontally compared to when the letters are read vertically. It has been shown that reading rate is similar for experienced adults who are trained to read either horizontal or vertical texts (Sun et al., 1985; see also Rayner, 1998). This implies that horizontal-vertical asymmetry in crowding is not necessarily a fixed characteristic but most likely it is experience- and skill-dependent.\n\nParticular attention should be given to the type of flankers used to induce crowding and contour interaction effects. We showed that when a surrounding box was placed at 2.5 stroke-widths (half a letter width) away from the target, the magnitude of crowding was not significantly different from that produced by either four letters or four bars, indicating that at this particular target-flanker separation, similar outcome would be found regardless of the flanker type. However, when placed at one stroke-width away from the target letter, flanking letters and bars caused approximately a further 10% reduction in performance to identify the target letter, compared to a surrounding box although this difference in performance did not reach statistical significance. Similar results have also been reported previously (Lalor et al., 2012). This may be of clinical implication since a number of crowded letter charts, designed to induce crowding, use a box as a crowding feature to surround the target letter (e.g. McGraw & Winn, 1993; Salt et al., 2007).\n\nVarious acuity tests designed to induce crowding typically used bars or other letters as their crowding feature, such as the logMAR crowded test, the Cambridge Crowding Cards and Sonksen LogMAR test. The separation between the target letter and the crowding feature typically is half an optotype width (but for the Sonsken logMAR test, the separation is one letter width). Formankiewicz & Waugh (2013) suggested that crowding in acuity tests could be enhanced by placing flankers closer to the target letter than in the currently available test. Our results complement this suggestion, where we also showed that larger magnitude of crowding can be obtained, at least for recognising single letters, if the crowding feature is placed at a distance closer than those designed in commercially available charts, specifically at one stroke-width away (0.2 letter width) from the target. At this particular separation, similar crowding magnitude was obtained with either letter or bar flankers. Indeed, larger foveal crowding effects have been reported for flankers that are placed closer than half a letter width away from an acuity target (Flom, 1991; Formankiewicz & Waugh, 2013; Lalor et al., 2016; Norgett & Siderov, 2014). Therefore, this separation could be considered in future designs of clinical acuity charts, so that these charts could be more sensitive in detecting eye conditions that suffer from crowding such as amblyopia. However, a surrounding box at this same separation produced approximately 10% less reduction of performance compared to other types of flankers. It has been reported that crowding reduces when flankers are grouped together, possibly aiding target recognition (for example, extending bars to form a surrounding box) (Banks & White, 1984; Manassi et al., 2012; Sayim et al., 2011).\n\nIn this study, abutting condition consistently produced the maximum amount of crowding, regardless of the flanker number or type. However, one must be careful when considering it in the design of clinical acuity charts. It has been observed that performance to localise the gap of a Landolt C was better when flankers abutted the target, compared to when the flankers were about 1 or 2 stroke widths away (Danilova & Bondarko, 2007; Flom et al., 1963). However, it was also reported that abutting flankers elicited the largest crowding magnitude compared to other target-flanker separations for localising the gap of a square C (Hairol et al., 2013) and for acuity measured with picture and letter optotypes (Formankiewicz & Waugh, 2013). Abutting may be a special case of crowding. When flankers and the target are just abutting, this combination may change the overall appearance of the target letter. It has been shown that dioptric blur reduces the amount of crowding obtained in abutting condition (Formankiewicz & Waugh, 2013) which would predict that those with uncorrected refractive error (or perhaps anisometropic amblyopia) would perform better than those with normal vision. Hence realistically, the spacing that exerts crowding is preferably not when the flankers are touching target.\n\nRecognition of letters in foveal viewing is influenced by the distance, type and number of flankers surrounding them. When a letter is surrounded by only two letter flankers, crowding magnitude differ significantly, depending whether they are in vertically- or horizontally-arranged configurations. This effect is possibly due to imprecise eye movements when extracting the target from the flankers, which could be experience- and skill-dependent. The magnitude of crowding is similar for a target surrounded by flankers at all four sides, regardless of the flanker type, at a target-flanker separation of half optotype width, which is widely used in commercially available crowded clinical charts. Maximum crowding can be induced at target-flanker separation of one stroke width (equivalent to 0.2 optotype width) when the target is surrounded on all four sides by either bars or other letters. A box however produces slightly less crowding effect, although not statistically significant, at this particular target-flanker separation. Our results have implications on the future designs of acuity tests that aim to exert the maximum crowding effect, so that they become more sensitive in detecting certain eye conditions such as amblyopia.\n\nF1000Research: Dataset 1. Percent correct performance for target identification, 10.5256/f1000research.8572.d120563 (Hairol et al., 2016).\n\nWritten informed consent for publication of their clinical data was obtained from the participants.",
"appendix": "Author contributions\n\n\n\nMIH and SK conceived the study. MIH designed the experiments with contributions from QMO, and QMO carried out the experiment. MIH prepared the first draft of the manuscript and QMO and SK contributed in subsequent drafts. 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\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank the staff of Optometry Clinic, Faculty of Health Sciences, UKM for their co-operation during the conduct of this study.\n\n\nReferences\n\nAtkinson J, Anker S, Evans C, et al.: Visual acuity testing of young children with the Cambridge Crowding Cards at 3 and 6 m. Acta Ophthalmol (Copenh). 1988; 66(5): 505–508. 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[
{
"id": "14036",
"date": "06 Jun 2016",
"name": "Yvonne Norgett",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 and accurately reflect the content of the article. Study design and methodology are sound and reflect the aims of the study. The conclusions are valid.\nSome minor points for correction: Introduction p.2- the author should correct the references to the 2 papers authored by Norgett and Siderov. The 2014 paper did not use commercially available tests and the 2011 paper did not have adult participants. Methods Please confirm if the participants wore their spectacle correction during the trials. Statistics p.3. spelling error 'Bonferroni'. In the last complete sentence on p.3, the sentence would read better with the word 'from' substituted with 'than', to read '...performance with top and bottom bars was significantly lower than unflanked conditions...' Table 1: The central column should have units- (letter-widths). Decimal places within the table should be consistent. Discussion spelling error in the last line of p.7- Sonksen First sentence of p.8 'currently available test' should be 'tests' plural. First sentence on 2nd column should read 'crowding magnitude differs' rather than 'differ'.",
"responses": []
},
{
"id": "14037",
"date": "04 Jul 2016",
"name": "Seong Taek Jeon",
"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\nReviewer’s General Summary The current research investigated the effect of type (bars vs. optotype) and position (top-and-bottom vs. side-by-side vs. all four sides) of flankers on foveal crowding (It was not clear if the surrounding box was a separate factor or level within the type or position). The aims of the study were two-folds: the authors tried to (1) compare how the above-listed factors affect the foveal crowding in terms of performance change relative to the reference uncrowded acuity, which is individually determined to yield 80 to 90% correct identification. (2) find the optimum separation between target and flankers that produce maximum crowding, where they found the optimal separation to be the abutting condition, not the single stroke-width, regardless of flanker type as well as position.\nOverall, the effect of flanker positions was presented in a series of figures by each flanker type, where the effect of crowding was greatest with four bars, followed by the left-and-right bars with top-and-bottom bars showing the minimum crowding (Figure 2). Similarly, the effect of crowding was maximised when all four sides of the target were surrounded by optotypes. However, crowding was minimal with left-and-right optotypes while the effect of crowding with the top-and-bottom condition was tantamount to that in four surrounding optotypes, which is different from when bars were used as flankers (Figure 3). The effect of surrounding box was compared to different flanker types only with four sides conditions, where the effect of crowding among the surrounding bars, optotypes, and box was more or less similar (Figure 4).\nMore specific comments can be found below. The reviewer recommends the paper to be accepted with the reservation that the authors address the issues in the specifics satisfactorily.\n\nMethod & Procedure\nWhat were the size of the monitor and its pixel resolution?\n\nGiven the unusually long viewing distance (11 to 13 metres), I gather the experiments were conducted in some sort of long hallway. Please describe the place and lighting condition with overall luminance level.\n\nMissing age and gender information of the participants.\n\nIn Figure 1, sample stimulus pictures are not correctly scaled.\n\nIn Figure 2-4 what are the error bars representing?\n\nIt is strongly recommended the authors provide the individual viewing distance and threshold letter size in visual angle along with the relative performance.\n\nFor the uncrowded performance, I am wondering if the authors tried any item analysis for each individual letters to see how each letter fared across observers. It has been shown (e.g., Bernard & Chung, 2011; Hanus & Vul, 2013; Liu & Arditi, 2001) that there are some differences in detectability among letters. This came to the reviewer’s attention due to the possible imbalance in the current experimental design, which is detailed below.\n\nIt was not clear to me if the surrounding box condition was treated as a separate factor or a level under flanker configuration or flanker type. This is critical since it is directly relevant to your analyses to follow. If I am not mistaken, the total number of conditions tested is 37, which is calculated from the combination of 6 target-to- flanker separations X 3 flanker positions (top-to-bottom vs.side-by-side vs. all four sides) X 2 flanker features/types (bars vs. optotypes) and an extra condition\n\nwith a surrounding box flanker. With this number of conditions, I am confused how you can possibly counterbalance the conditions with seven letters to be equally presented across conditions in both the single letter and crowding condition when the total numbers of trials used were 400 and 3200 respectively. This problem still holds even when I include the surrounding box condition as a level under either flanker feature or position. Together with the possible difference in legibility among the letters, this imbalance can be a serious threat to the validity of the study.\n\nResults & Analyses\nAccording to the results, maximum crowding occurs when flankers touch the target regardless of flanker types and positions then the magnitude of crowding monotonically diminishes as a function of target-flanker separation. Especially for the bar flankers, this is somewhat different from the literature and the reviewer’s personal experience because bar flankers do not crowd some targets anymore when bars are blended with features of certain letters. For example, when bars are touching the sides to the vertical strokes of an H as in Figure 1(a) or top and bottom to an upright T as in Figure 1(b), they become a distinct featural addition to a new amalgamated pattern that can be readily picked up by foveal vision. This in turn may facilitate the identification of the target, which has been demonstrated many times as a dipper curve, instead of smooth and monotonic curve, when performance is plotted as a function of separation (Bedell et al., 2013; Danilova and Bondarko, 2006; Flom, Weymouth, and Kahneman, 1963; Jeon et al., 2010; Simmers et al., 1999). Thus, the monotonic function of crowding shown in the current paper begs explanation or discussion with respect to previous literature.\n\nIn relation to the imbalance issue mentioned above, it was not clear whether a single omnibus ANOVA or multiple ANOVAs were performed. Furthermore, it is still not clear if the surrounding box condition was treated as a factor or a level. Please make sure to explain clearly how the surrounding box condition was treated in the analysis.\n\nGiven the consistent trend in data regardless of flanker type and position where the relative performance monotonically increases as a function of flanker-target separation, a generalised linear modelling with nested model testing seems more appropriate than ANOVA. It will provide a more structured account on how the effect of flanker types and positions changes with respect to separation.\n\nDiscussion\nAs mentioned in the last paragraph of the introduction that the results would have an application in designing a clinical letter test, I expected in-depth discussion of how the current results can be compared to the existing clinical crowding acuity tests such as Cambridge Crowding Cards (Atkinson et al., 1988) or Glasgow Acuity Cards (McGraw and Winn, 1995). A few insightful recommendations or suggestions on this matter seem to be a nice addition to the current section.\n\nIn the 1st sentence of the last paragraph, the authors are saying, “In this study, abutting condition consistently produced the maximum amount of crowding, regardless of the flanker number or type,” which is not consistent with the abstract where they said, “Flankers abutting the target and at separation of one stroke width caused the largest reduction in performance, regardless of flanker type.”\n\nTypos Abstract line 13: ad > and pg. 3 under Statistics: Benferroni > Bonferroni",
"responses": []
}
] | 1
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https://f1000research.com/articles/5-1013
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https://f1000research.com/articles/5-1010/v1
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26 May 16
|
{
"type": "Review",
"title": "Non-ionotropic signaling by the NMDA receptor: controversy and opportunity",
"authors": [
"John A. Gray",
"Karen Zito",
"Johannes W. Hell"
],
"abstract": "Provocative emerging evidence suggests that the N-methyl-D-aspartate (NMDA) receptor can signal in the absence of ion flux through the receptor. This non-ionotropic signaling is thought to be due to agonist-induced conformational changes in the receptor, independently of channel opening. Non-ionotropic NMDA receptor signaling has been proposed to be sufficient to induce synaptic long-term depression (LTD), directly challenging the decades-old model that prolonged low-level calcium influx is required to induce LTD. Here, we briefly review these recent findings, focusing primarily on the potential role of non-ionotropic signaling in NMDA receptor-mediated LTD. Further reports concerning additional roles of non-ionotropic NMDA receptor signaling are also discussed. If validated, this new view of NMDA receptor-mediated signaling will usher in an exciting new era of exploring synapse function and dysfunction.",
"keywords": [
"NMDA",
"Non-ionotropic",
"signaling",
"LTD"
],
"content": "Introduction\n\nN-methyl-d-aspartate receptors (NMDARs) are glutamate-gated cation channels that play crucial roles in neurodevelopment and bidirectional synaptic plasticity. Most of the functions of the NMDAR have been attributed to the influx of calcium ions during channel opening1–10. However, a flurry of recent studies11–18 have provided more systematic support for earlier studies19–22 suggesting that agonist binding to NMDARs can transmit information to signaling molecules independently of Ca2+ influx through the channel. If confirmed, these findings may lead to new pharmacological approaches to target specific synaptic signaling cascades in the numerous disorders attributed to synaptic dysfunction, including autism, schizophrenia, post-traumatic stress disorder, epilepsy, addiction, and Alzheimer’s disease.\n\nSynaptic plasticity is a well-established cellular model for learning and memory and involves the persistent increase (long-term potentiation, or LTP) and weakening (long-term depression, or LTD) of synaptic strength in response to various patterns of activity. At most excitatory synapses in the brain, NMDAR activation is important for the induction of both LTP and LTD23–26. The widely accepted model for how activation of a single receptor can produce opposite changes in synaptic strength involves the amount and duration of Ca2+ influx27–30. This model posits that brief periods of high-frequency synaptic activity lead to a large, rapid increase in intracellular Ca2+ through the NMDAR that activates a series of biochemical steps, leading to LTP31. Conversely, prolonged periods of low-frequency synaptic activity drive a modest increase in Ca2+ through NMDARs that activates a different series of biochemical steps, leading to LTD24,25,32.\n\nFor NMDAR-dependent LTD, the model that a modest increase in intracellular Ca2+ is necessary for LTD induction has recently been challenged. Using systematic pharmacological approaches that block ion flux but spare glutamate binding to the NMDAR, Nabavi et al.15 found that LTD could still be induced in an NMDAR-dependent manner. Here, we will review this provocative finding and follow-up studies, highlighting a direct refutation33 and confirmation16 of this result. Furthermore, we will discuss possible explanations for the disparate findings and additional reports of NMDAR-mediated signaling independent of channel opening. Given the importance of NMDARs in synaptic development and plasticity, these findings have the potential to be transformative but need further detailed and rigorous follow-up.\n\n\nDoes NMDAR-dependent LTD involve non-ionotropic mechanisms?\n\nNMDARs have a rich and complex pharmacology. Most NMDARs are heterotetramers containing two GluN1 subunits and two GluN2 subunits. One of the notable properties of NMDARs is that they are blocked by magnesium ions at resting membrane potentials35; opening of NMDARs requires simultaneous activation by glutamate and depolarization to relieve the Mg2+ block. In addition, NMDARs are unique among neurotransmitter receptors in having an absolute requirement for the binding of a co-agonist in addition to glutamate in order to open the channel. Glutamate binds to the extracellular ligand-binding domain on the GluN2 subunits, whereas the co-agonist, which is either glycine or d-serine, binds to the homologous ligand-binding domain on the GluN1 subunits (Figure 1).\n\nMost NMDARs are tetrameric proteins containing two GluN1 and two GluN2 subunits (for clarity, only one of each is pictured). For the NMDAR channel to open, both glutamate and a co-agonist, which can be glycine or d-serine, need to bind to clamshell-like ligand-binding domains on the GluN2 and GluN1 subunits, respectively. There are multiple approaches to block ion flow through the NMDAR channel: a competitive antagonist for the glutamate-binding site on the GluN2 subunits (e.g. AP-5), a competitive antagonist for the glycine/d-serine binding site on the GluN1 subunits (e.g. 7-CK), or an uncompetitive blocker of the channel itself (e.g. MK-801). 7-CK, 7-chlorokynurenic acid; AP-5, (2R)-amino-5-phosphonovaleric acid; MK-801, dizocilpine.\n\nThere are multiple approaches to blocking ion flow through the NMDAR: (1) a competitive antagonist of the glutamate-binding site on GluN2 (e.g. AP-5), (2) a competitive antagonist of the co-agonist site on GluN1 (e.g. 7-chlorokynurenate, or 7-CK), or (3) an uncompetitive blocker of the channel pore (e.g. MK-801) (Figure 1). Using each of these pharmacologic strategies to block ion flow through the NMDAR, Nabavi et al.15 surprisingly found that only the glutamate site antagonist AP-5 blocked LTD, suggesting that glutamate binding is required for LTD but not co-agonist binding or ion flow through the NMDAR. Although metabotropic glutamate receptor (mGluR)-mediated forms of LTD can also occur at these synapses36–38, application of an mGluR5 inhibitor and an inhibitor of L-type Ca2+ channels (which are required for mGluR1-mediated LTD39) did not block the LTD observed in the presence of 7-CK or MK-801, supporting the notion that signaling through mGluRs did not provide an alternate source for a rise in intracellular Ca2+ leading to LTD.\n\nSo, what about the two decades of evidence that support a fundamental role for NMDAR-mediated Ca2+ influx in LTD32? Indeed, introducing Ca2+ chelators intracellularly to the post-synaptic neuron prevents LTD24,40–43. In addition, increases in intracellular Ca2+ levels through activation of voltage-gated calcium channels44 or photolysis of caged Ca2+30,45 induces synaptic depression that occludes additional LTD. Furthermore, LTD requires signaling mechanisms which are dependent on Ca2+, including the activation of calcineurin46 and hippocalcin47. Nabavi et al.15 also saw that Ca2+ chelation inhibited LTD, but they proposed that the reduction in basal intracellular Ca2+ concentration by strong chelation is responsible for the loss of LTD. To test this idea, they buffered the intracellular Ca2+ concentration with the strong chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) along with additional free Ca2+ in order to prevent acute rises in Ca2+ while maintaining baseline Ca2+ near physiological levels. This clamping of Ca2+ at baseline levels did not block LTD, suggesting that low levels of basal Ca2+, but not acute elevations of Ca2+ through the NMDAR, are required for LTD. Remarkably, when Ca2+ influx was blocked through the NMDAR with MK-801, a high-frequency stimulus that normally induces LTP actually resulted in LTD. Together, these results support that NMDAR-dependent LTD requires glutamate binding to the NMDAR but not Ca2+ influx.\n\nThe surprising and provocative work by Nabavi et al.15 was soon directly challenged. Specifically, Babiec et al.33 found that, in contrast to Nabavi et al., MK-801 effectively blocked LTD induction in slices from both young and adult animals. Furthermore, they showed that MK-801 blocked a chemical form of LTD induced by bath application of the glutamate site agonist NMDA. In addition, lowering extracellular Ca2+ concentration also blocked LTD, supporting previous studies defining the role of Ca2+ in LTD32. These results stand in direct conflict with those of Nabavi et al., and the reasons for these inconsistent findings remain unclear (see below for additional discussion).\n\nOthers have begun to weigh in. Recently, Stein et al.16 examined the role of non-ionotropic NMDAR signaling in activity-induced dendritic spine shrinkage, a structural correlate of LTD. Using two-photon glutamate uncaging and time-lapse imaging, they found that low-frequency uncaging led to spine shrinkage even when the NMDAR glycine/d-serine site was blocked with 7-CK. Furthermore, the presence of 7-CK or MK-801 converted high-frequency uncaging-induced spine enlargement to spine shrinkage, similar to the findings by Nabavi et al. for LTD. This spine shrinkage evoked in the presence of 7-CK was not inhibited by co-application of antagonists of mGluR1 and mGluR5. Importantly, since two-photon glutamate uncaging was used to bypass presynaptic neurotransmitter release, the potential effects of the pharmacological agents on pre-synaptic NMDARs were avoided. These results further support the idea that non-ionotropic NMDAR-mediated signaling mechanisms can drive synaptic depression.\n\nWhy the inconsistent results among laboratories48? It is not yet clear. One possibility is that ionotropic and non-ionotropic mechanisms coexist. Alternatively, there may be unrecognized experimental differences (for example, in slice preparation, solutions, timing of drug application and removal, perfusion, and temperature). Indeed, a major limitation of these studies is the reliance on pharmacology. In most previous studies, AP-5 or other competitive glutamate site antagonists are used to block NMDAR activity, and we are unaware of any published examples in which AP-5 did not block NMDAR-mediated LTD. The examination of glycine co-agonist site antagonists in LTD, however, is a newer development, and it would have been enlightening if Babiec et al. had included one in their analysis. 7-CK is an early derivative of the naturally occurring kynurenic acid, which is a non-selective antagonist of all ionotropic glutamate receptors as well as the α7-nicotinic receptor. Although 7-CK is more potent and selective than kynurenic acid, it still exhibits a significant blockade of other receptors49. Follow-up studies using higher-potency glycine site antagonists, and ones of different chemical classes, such as MDL 105,51950, will be of key importance.\n\nMost of the conflicting results described above involve MK-801, an uncompetitive open-channel blocker of NMDARs51. Oddly enough, it appears that this controversy is not new. Previous studies have shown an inhibition of LTD by MK-801 in the CA1 region of the hippocampus52,53 and the mouse visual cortex54. However, an older report showed that although MK-801 blocked LTP, it did not block low-frequency stimulation-induced LTD in the hippocampus19. Perhaps others have observed a lack of LTD inhibition by MK-801 but attributed this to experimental error or incomplete NMDAR blockade and therefore never reported it. Indeed, because of the use-dependent nature of MK-801, incomplete blockade of synaptic NMDARs may allow small local increases in Ca2+ during repetitive stimulation right at the channel mouth that is not effectively buffered. Although this explanation is unlikely, it is difficult to rule out. Indeed, in an attempt to control for this use dependence of MK-801, slices are often incubated for a few hours in order to allow spontaneous activity to block all NMDARs prior to the onset of stimulation15,33, although others find that LTD is blocked when MK-801 is applied just minutes before induction55–57. Another issue is that the current tools for measuring changes in intracellular Ca2+ are not sensitive enough to detect trace amounts of influx through the NMDAR, especially if that Ca2+ is immediately bound to proteins within the NMDAR signaling complex. This could possibly be examined with a Ca2+-impermeable or channel “dead” NMDAR or by attaching a genetically encoded Ca2+ sensor directly to the NMDAR intracellular domains or associated proteins.\n\n\nAgonist-induced conformational changes in the NMDAR intracellular domains\n\nOf course, the possibility of non-ionotropic signaling by NMDARs requires evidence of conformational changes upon agonist binding. While perhaps surprising for ligand-gated ion channels, non-ionotropic signaling is extremely common. G-protein-coupled receptors (GPCRs) comprise the largest protein superfamily in mammalian genomes and act solely through conformational changes upon extracellular agonist binding58,59. Indeed, the β2-adrenergic receptor, a prototypical GPCR, has only 168 intracellularly located amino acids, whereas NMDARs with their tetrameric structure and long complex C-terminal tails can have upwards of 1700 intracellular residues. In addition, at the post-synaptic density, NMDARs are a central member of a large macromolecular complex comprising signaling molecules, scaffolding and adaptor proteins, and cytoskeletal proteins60,61. Through these complex interactions, NMDARs are in a key position to engage and regulate intracellular signaling machinery. Indeed, while the long C-terminal tails of NMDARs have been presumed to be intrinsically unstructured, the complex scaffolding and interactions at the post-synaptic density may impart the secondary and tertiary structure62 required to transmit information via agonist-induced conformational changes63.\n\nRecently, Dore et al.13 demonstrated that NMDA binding to the glutamate site of the GluN2 subunits drives conformational changes in the NMDAR intracellular domains. Specifically, either green fluorescent protein (GFP) or mCherry was fused to the C-terminal tails of GluN1 subunits, and primary hippocampal neurons were co-transfected with both GFP- and mCherry-containing GluN1 subunits. Importantly, although the GluN2 subunits contain the glutamate-binding domain, GluN1 was chosen because tagging GluN2 subunits affects their trafficking and synaptic targeting64. They then used fluorescence lifetime imaging microscopy (FLIM) to measure the lifetime of GFP fluorescence, which is reduced when in close proximity to mCherry because of Förster resonance energy transfer (FRET)65. They found that NMDA caused rapid changes in GFP fluorescence lifetime in the presence of 7-CK or MK-801, but not in the presence of AP-5, providing evidence for agonist-induced, but ion flow-independent, conformational changes in the NMDAR C-terminal tails. In an accompanying study using similar techniques, Aow et al.11 showed that NMDA binding, even in the presence of 7-CK (but not AP-5), leads to changes in the interactions between GluN1-GFP and various signaling proteins known to associate with the NMDAR which were tagged with the FRET acceptor mCherry. Specifically, they measured a rapid transient change in the interaction between GluN1 and protein phosphatase 1 (PP1) and a delayed but persistent change in the interaction of GluN1 with calcium/calmodulin-dependent protein kinase II (CaMKII). Together, these findings provide support that agonist binding to the NMDAR can produce intracellular conformational changes independently of ion flow through the receptor channel. These conformational changes are consistent with non-ionotropic signaling following glutamate binding to NMDARs.\n\n\nOther non-ionotropic NMDAR signaling\n\nIn addition to LTD, other studies have suggested additional roles for non-ionotropic NMDAR signaling. In Alzheimer’s disease, for example, impaired hippocampal synapse dysfunction is an early event66,67 that is associated with increased levels of diffusible oligomeric assemblies of the amyloid-beta (Aβ) protein68–70. These Aβ oligomers cause a rapid synaptic depression that is dependent on NMDAR activity71–75. Recently, it has been shown that this NMDAR-dependent, Aβ-induced synaptic depression does not require ion flux though the channel12,14,17. Kessels et al.14 found that increasing Aβ levels in organotypic hippocampal slice cultures through the viral expression of the β-secretase product of the amyloid precursor protein leads to a baseline synaptic depression that can be blocked by AP-5, but not 7-CK or MK-801. Similarly, Tamburri et al.17 found that this synaptic depression can occur in acute hippocampal slices within 15 minutes of perfusion of oligomeric Aβ and that this rapid depression was dependent on NMDAR activity and was blocked by AP-5, but not MK-801, again suggesting that it did not require ion flux through the receptor17. In addition to Aβ-induced synaptic depression, Aβ oligomer-induced synapse loss was recently demonstrated to be blocked by AP-5, but not MK-80112. Together, these results suggest that non-ionotropic NMDAR signaling contributes to the Aβ-induced synaptic dysfunction in Alzheimer’s disease and may suggest a common mechanism between Aβ-induced synaptic depression and non-ionotropic NMDAR-dependent LTD.\n\nOther studies have described non-ionotropic NMDAR signaling, providing additional support for its physiological importance. In the earliest of these studies, glutamate binding was shown to induce dephosphorylation of the GluN2A subunit, resulting in the endocytosis of the receptor in the absence of ion flux21. In another, co-activation of NMDARs and mGluR5 led to extracellular signal-regulated kinase (ERK) activation and increased c-Fos expression independent of ion flux but dependent on the interaction of the GluN2 C-terminal tail with scaffolding proteins in the post-synaptic density22. NMDAR activation is also central to pathological processes that lead to neuronal death and non-ionotropic NMDAR-mediated signaling through Src kinase, and pannexin-1 was recently reported to occur during excitotoxicity18. In addition to glutamate, co-agonist binding to the glycine site on the GluN1 subunits may also be involved in non-ionotropic signaling. For example, glycine or D-serine binding has been found to prime the NMDAR for subsequent clathrin-mediated endocytosis in the presence of AP-5 but not glycine site antagonists20.\n\n\nA new horizon for NMDAR biology?\n\nHere we have reviewed the current literature suggesting that activation of NMDARs can activate intracellular signaling independent of ion flux through the receptor. These results have been quite provocative, though from our perspective not entirely unexpected as NMDAR are part of a large multi-protein complex at the post-synaptic density and thus are in an ideal position to have conformation-based signaling. Although the physiological significance of potential parallel ionotropic and non-ionotropic NMDAR signaling processes remains controversial, their coexistence predicts the possibility for divergent signaling events based on agonist and co-agonist availability, channel opening, and receptor subunit composition. Ultimately, further exploration of this model may open a new frontier of NMDAR biology and lead to the development of novel approaches for targeting NMDAR signaling for the treatment of multiple neuropsychiatric disorders.\n\n\nAbbreviations\n\n7-CK, 7-chlorokynurenic acid; Aβ, amyloid-beta; AP-5, (2R)-amino-5-phosphonovaleric acid; FRET, Förster resonance energy transfer; GFP, green fluorescent protein; GPCR, G-protein-coupled receptor; LTD, long-term depression; LTP, long-term potentiation; mGluR, metabotropic glutamate receptor; MK-801, dizocilpine; NMDA, N-methyl D-aspartate; NMDAR, N-methyl D-aspartate receptor.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nJohn A. Gray is supported by a NARSAD Young Investigator Award from the Brain and Behavior Research Foundation and by the National Institutes of Health (NIH) (K08 MH100562 and a pilot through P30 AG010129). Karen Zito is supported by the NIH (R01 NS062736). Johannes W. Hell is supported by the NIH (R01 NS078792, R01 AG017502, and R01 MH097887).\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\nCole AJ, Saffen DW, Baraban JM, et al.: Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation. Nature. 1989; 340(6233): 474–6. PubMed Abstract | Publisher Full Text\n\nHardingham GE, Arnold FJ, Bading H: A calcium microdomain near NMDA receptors: on switch for ERK-dependent synapse-to-nucleus communication. Nat Neurosci. 2001; 4(6): 565–6. PubMed Abstract | Publisher Full Text\n\nHayashi Y, Shi SH, Esteban JA, et al.: Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science. 2000; 287(5461): 2262–7. PubMed Abstract | Publisher Full Text\n\nMayer ML, Westbrook GL, Guthrie PB: Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature. 1984; 309(5965): 261–3. PubMed Abstract | Publisher Full Text\n\nNägerl UV, Eberhorn N, Cambridge SB, et al.: Bidirectional activity-dependent morphological plasticity in hippocampal neurons. Neuron. 2004; 44(5): 759–67. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nShi SH, Hayashi Y, Petralia RS, et al.: Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science. 1999; 284(5421): 1811–6. PubMed Abstract | Publisher Full Text\n\nZhou Q, Homma KJ, Poo MM: Shrinkage of dendritic spines associated with long-term depression of hippocampal synapses. Neuron. 2004; 44(5): 749–57. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nVan Dongen AM, editor: Biology of the NMDA Receptor. Boca Raton, FL. 2009. PubMed Abstract\n\nMatsuzaki M, Honkura N, Ellis-Davies GC, et al.: Structural basis of long-term potentiation in single dendritic spines. Nature. 2004; 429(6993): 761–6. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nZito K, Scheuss V: NMDA Receptor Function and Physiological Modulation. In Encyclopedia of Neuroscience. Squire LR, Editor, Oxford: Academic Press. 2009; 1157–1164. Publisher Full Text\n\nAow J, Dore K, Malinow R: Conformational signaling required for synaptic plasticity by the NMDA receptor complex. Proc Natl Acad Sci U S A. 2015; 112(47): 14711–6. 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PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nStein IS, Gray JA, Zito K: Non-Ionotropic NMDA Receptor Signaling Drives Activity-Induced Dendritic Spine Shrinkage. J Neurosci. 2015; 35(35): 12303–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTamburri A, Dudilot A, Licea S, et al.: NMDA-receptor activation but not ion flux is required for amyloid-beta induced synaptic depression. PLoS One. 2013; 8(6): e65350. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeilinger NL, Lohman AW, Rakai BD, et al.: Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity. Nat Neurosci. 2016; 19(3): 432–42. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMayford M, Wang J, Kandel ER, et al.: CaMKII regulates the frequency-response function of hippocampal synapses for the production of both LTD and LTP. Cell. 1995; 81(6): 891–904. 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}
|
[
{
"id": "14026",
"date": "26 May 2016",
"name": "Isabel Pérez-Otaño",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14027",
"date": "26 May 2016",
"name": "Yasunori Hayashi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1010
|
https://f1000research.com/articles/5-1009/v1
|
26 May 16
|
{
"type": "Review",
"title": "Targeting the latent reservoir to achieve functional HIV cure",
"authors": [
"Daniele C. Cary",
"B. Matija Peterlin",
"Daniele C. Cary"
],
"abstract": "While highly active anti-retroviral therapy has greatly improved the lives of HIV-infected individuals, current treatments are unable to completely eradicate the virus. This is due to the presence of HIV latently infected cells which harbor transcriptionally silent HIV. Latent HIV does not replicate or produce viral proteins, thereby preventing efficient targeting by anti-retroviral drugs. Strategies to target the HIV latent reservoir include viral reactivation, enhancing host defense mechanisms, keeping latent HIV silent, and using gene therapy techniques to knock out or reactivate latent HIV. While research into each of these areas has yielded promising results, currently no one mechanism eradicates latent HIV. Instead, combinations of these approaches should be considered for a potential HIV functional cure.",
"keywords": [
"HIV",
"latent HIV",
"targeting latent HIV",
"viral reactivation",
"host defense mechanisms",
"gene therapy"
],
"content": "Introduction\n\nIn the twenty years since the implementation of highly active anti-retroviral therapy (HAART), the overall face of HIV as a global health issue has changed1. HAART—composed of a cocktail of anti-retroviral drugs which target proteins expressed at different steps in the HIV replication cycle—can affect only cells that harbor actively replicating virus. HIV+ individuals are able to live fairly normal lives on maintenance HAART, with minimal side effects. Nevertheless, the effects of HIV infection continue to be evident in these suppressed individuals, who continue to suffer from a number of metabolic, immunologic, and neurologic co-morbidities2. Thus, despite reducing plasma viremia below detection limits, the virus is not eliminated. There is evidence that low levels of replication occur in suppressed individuals, primarily in tissue reservoirs; however, this is not reflected in systemic plasma viremia in these individuals3,4. HAART requires life-long administration. Following even brief treatment interruption, HIV rebounds rapidly from its reservoirs5–7. Goals of the present research are to eliminate, suppress permanently, or render cells inhospitable to the hidden HIV in infected individuals.\n\nResearch efforts to understand and target HIV reservoirs have focused on four main categories outlined in this review (Figure 1): first, reactivation of latent HIV by capitalizing on the ability of host cellular activation signals and transcription factors (TFs) to ‘shock’ the virus out of hiding; second, killing of reactivated HIV by strengthening the immune system, which has been crippled by the infection; third, keeping latent reservoirs permanently suppressed; and, finally, targeting HIV and CD4+ T cells, which are the primary host cells for the virus, via new gene therapy approaches.\n\nFour research areas, which reactivate HIV (1. shock), eliminate HIV (2. kill), silence HIV (3. silence), or alter the immune system to resist HIV (4. gene therapy) should contribute to the functional or complete cure of HIV in infected individuals. Within each area are individual components of that therapy. They can be applied individually or in combinations, which should decrease their doses and deleterious effects. Most likely, there will be additional approaches in the future.\n\n\nShock\n\nChronic infection by HIV is characterized by severe depletion of CD4+ T cells and continuing inflammation, which contribute to HIV-associated co-morbidities2. Continued exposure to inflammatory cytokines exhausts the immune system. It also elevates the expression of the receptors programmed death 1 (PD-1)8 and cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4)9 on T cells. Blockade of these molecules is used as a treatment for solid tumors10 and could reinvigorate exhausted T cells in HIV+ patients11. These individuals also produce elevated levels of inhibitory cytokines interleukin (IL)-10 and transforming growth factor–beta (TGF-β)12,13. Indeed, blocking IL-10 results in increased T cell activity in a hepatitis C infection model14,15.\n\nGrowth factor therapy, including treatment with IL-2, -7, or -15, is being explored as a means to stimulate T cell recovery. IL-2 and IL-7 are important T cell growth and proliferation factors. Infusion with IL-2 and IL-7 results in enhanced T cell production and memory T cell proliferation16–18. IL-15 enhances cytotoxic CD8+ T lymphocyte (CTL) and natural killer (NK) cell activity in vitro. Indeed, the IL-15 super-agonist ALT-803 is currently in preclinical trials19.\n\nLatent HIV is primarily found in resting CD4+ T cells in the periphery. Resting cells have low levels of cellular TFs, which are also required for HIV replication, including NF-κB, P-TEFb, and CDK1120,21. Among the first examined latency reversing agents (LRAs) were histone deacetylase inhibitors (HDACis) and BET bromodomain inhibitors (BETis), which induce chromatin stress and induce the release of positive transcription elongation factor b (P-TEFb) from its repressive complex22. HDACis—such as panobinostat23, romidepsin24, SAHA25, and valproic acid26—and BETis—such as JQ127—all reactivate HIV in cell line models of latency. However, they do not work in human primary resting infected T cells28,29 because they contain very low levels of necessary TFs20,21. Thus, clinical trials with SAHA resulted in only a modest and transient reactivation of HIV30, making it an impractical mono-therapy for HIV reactivation.\n\nSince HDACis and BETis do not increase levels of required TFs, some activation of CD4+ T cells is required. Indeed, protein kinase C (PKC) agonists, such as prostratin31 and bryostatin32, and the MAPK agonist procyanidin33,34 can reactivate HIV in cell line models and primary CD4+ T cells. However, prostratin is toxic at therapeutic levels, leading to muscle pain, respiratory distress, and hypertension. Bryostatin, derived from a marine animal, Bugula neritina, not only has similar side effects but is also cost prohibitive to manufacture. Because of these limitations, a number of synthetic analogues of prostratin and bryostatin with reduced toxicity in vitro are being developed35–37. Ingenols, which are purified from Euphorbia plants, represent additional PKC agonists of interest. Native and chemically modified ingenols reactivate HIV in cell lines and primary T cells38–40. These PKC agonists also increase cellular levels of necessary TFs38. Thus, select MAPK and PKC agonists represent attractive candidates to reactivate latent HIV.\n\nCombining several of these approaches has the greatest potential to purge the viral reservoir. Indeed, lower doses of a T cell activator and an LRA (HDACi or BETi) can be administered for increased potency and reduced pro-inflammatory responses41–43. Further understanding of HIV integration, transcription, and reactivation, as well as host cell behaviors, will inform optimal combinations of activators and LRAs.\n\n\nKill\n\nStrategies to remove HIV by enhancing the killing by CTL and NK cells44 or via broadly neutralizing antibodies (bNAbs) represent the second major field of research in HIV eradication. It is also important to investigate kill strategies in the context of the aforementioned shock therapies because many of the treatments proposed to reactivate latent HIV also dampen CTL function45, which is already impaired in HIV+ individuals11.\n\nUsing modified cytomegalovirus (CMV), a live vaccine expressing several simian immunodeficiency virus (SIV) antigens, was found to protect rhesus macaques against viral challenge46–48. Vaccinated animals initially appeared to be infected; however, they gained protection against SIV and showed enhanced effector T cell function against viral antigens.\n\nAnother approach involves bNAbs49. Following infection, anti-HIV antibodies are abundant in HIV+ patients; however, owing to the ability of the virus to mutate, the majority of them fail to eliminate the virus. bNAbs are the exception, in that they recognize many clades of HIV as well as escape mutants of the virus. In several studies, they not only neutralized virions released from activated CD4+ T cells from patients50 but also reduced the viral rebound following HIV reactivation in a humanized mouse model51. However, even the most potent bNAbs are each only effective against a narrow subset of HIV clinical isolates, suggesting that effective bNAb approaches may require a combination of several bNAbs52. A second antibody approach utilizes bispecific antibodies, wherein one arm of the Fab portion of the antibody recognizes HIV envelope and the second arm recognizes CD3, making the cell vulnerable to CTL-mediated killing.\n\nFinally, in an effort to achieve more effective killing, chimeric antigen receptors (CARs), which increase T cell receptor avidity and activation, are being explored. They can be engineered to recognize specific viral proteins; CARs against CD19, which is a B cell receptor, led to an astounding 90% remission rate in acute leukemia53,54. However, one caveat to CARs is that these cells are long-lived and can have substantial off-target effects.\n\n\nSilence\n\nThe success of HAART has demonstrated that keeping the virus suppressed results in markedly healthier individuals. Resting infected cells do not produce HIV. Thus, these strategies rely on reducing T cell activation, which should also reduce the HIV-associated inflammation found in chronically infected individuals2. JAK and STAT molecules are important signaling molecules associated with many cytokine receptors. Ruxolitinib and tofacitinib, two JAK inhibitors that are approved for the treatment of rheumatoid arthritis and myelofibrosis, were tested against HIV, HIV2, and simian HIV (SHIV). They inhibited HIV reactivation55, and, furthermore, ruxolitinib attenuated encephalitis symptoms in infected humanized mice56. Cyclosporine A, an immunosuppressant used primarily to prevent transplant rejection57, inhibits T cell proliferation by blocking IL-2 signaling in T cells58. Infected patients treated with cyclosporine A had some T cell recovery59 but limited suppression of HIV replication60,61.\n\nThe inhibitor didehydro-cortistatin A (dCA) acts via a suppressive mechanism that primarily targets HIV transcription. dCA binds to the basic domain in the HIV regulatory protein Tat, inhibits its interactions with the RNA response element TAR, and prevents its activation of HIV transcription62. dCA inhibits HIV reactivation in cell lines, primary cells, and peripheral blood mononuclear cells (PBMCs) from HAART-suppressed patients62. Furthermore, dCA may also contribute to continued HIV suppression by inhibiting inflammatory cytokine expression63.\n\n\nGene therapy\n\nRecently, a number of groups have taken advantage of cutting edge gene therapy approaches to HIV cure. However, as with any gene therapy approach, the barriers include delivery, specificity, off-target effects, costs, and ethical concerns.\n\nThe single case of successful HIV cure was achieved by the reconstitution of the patient’s immune system with donor bone marrow containing a natural mutation in the CCR5 HIV co-receptor64. This patient was treated for acute leukemia with several courses of total lymphoid irradiation followed by two separate bone marrow transplantations. Attempts to replicate this therapy used the Zn++ finger nuclease65 and more recently CRISPR/Cas9 targeting of CCR5 to induce the delta 32 mutation in patients’ own hematopoietic cells66,67, which were then returned to the host. Since only mature cells were used, the effects of these manipulated cells were not permanent65. Recent work using CRISPR/Cas9 to target the second HIV co-receptor, CXCR4, has also yielded promising results68,69.\n\nWhile HIV and SIV are highly related viruses, HIV cannot infect non-human primates, as their restriction factors block HIV infection more effectively than their human counterparts70. Therefore, altering human restriction factors to behave like their simian counterparts represents an attractive strategy. One such factor is TRIM5. Of special interest is TRIM5 from owl monkeys, which is linked in frame to cyclophilin A, and this fusion protein blocks HIV71. Using lentiviral vectors to deliver Trim-Cyp has blocked HIV effectively in cell lines and primary T cells72. Additionally, it has been used successfully in a triple combination anti-HIV lentiviral vector approach in an infected humanized mouse model73.\n\nRecently, CRISPR/Cas9 technology has emerged as the most versatile and effective gene therapy approach. Using a DNA targeting strategy utilized by bacterial CRISPR, any number of specific guide RNAs can be loaded into the Cas9 protein to target specific areas of DNA for knock out or knock in of genes74. Similarly, this technology has been used to knock out and reactivate latent HIV. Targeting various regions of the HIV LTR inactivated the virus in infected cell lines75 and prevented their reinfection76. However, viral target sequences can mutate, and HIV LTR-specific guide RNA can fail to recognize and target the mutant sequences, preventing long-term eradication by this method77. To reactivate HIV, a defective Cas9 protein (dCas9) is used, which is fused to four copies of the herpes simplex VP16 activation domain (VP64) or a synergistic activation mediator (SAM) complex. Again, guide RNAs bring these dCas9 activators to the initiated transcription machinery. This targeting results in potent reactivation in latently infected cell lines78–80.\n\n\nSummary\n\nAlthough HIV infection in the era of HAART has become a manageable chronic infection, problems with adherence to drug regiment, co-morbidities, and the emergence of drug resistance emphasize the need for continued research into HIV cure. Since the barrier to cure is the HIV reservoir, targeting this persistent virus is critical. The approaches detailed in this review represent a spectrum of the current research: however, eliminating the remaining 106 to 108 latently infected cells81 will require a combination of approaches. Mechanisms, such as HIV reactivation, will reveal hidden virus. However, the severely crippled immune system and further decreased CTL function indicate that it must be paired with the boosting of anti-viral host defenses. Likewise, keeping latent HIV in a suppressed state could keep HIV+ patients relatively healthy but less able to resist other infections and/or cancer. Using gene therapy to create a parallel immune system, where cells resist HIV infection, could complement all other approaches but is not scalable or affordable in resource-poor countries. While none of these approaches represent the eradication of HIV, combining several treatment modalities could bring us closer to a functional cure, where prolonged HAART-free and disease-free intervals would be achieved in infected patients.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work was supported by National Institutes of Health grants U19 AI096113 (Martin Delaney Collaboratory of AIDS Researchers for Eradication [CARE] Center Grant, David Margolis, PI), P50 GM082250 (HARC Center Grant, Alan Frankel and Nevan Krogan, coPIs) and AI1049104 (to B.M.P.).\n\n\nAcknowledgements\n\nWe thank Koh Fujinaga and Wei Shao for helpful discussions.\n\n\nReferences\n\nUNAIDS: AIDS by the numbers 2015. Joint United Nations Programme on HIV/AIDS (UNAIDS). 2015. Reference Source\n\nMarin B, Thiébaut R, Bucher HC, et al.: Non-AIDS-defining deaths and immunodeficiency in the era of combination antiretroviral therapy. AIDS. 2009; 23(13): 1743–53. 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Curr HIV Res. 2010; 8(6): 418–29. PubMed Abstract | Publisher Full Text\n\nHori T, Barnor J, Huu TN, et al.: Procyanidin trimer C1 derived from Theobroma cacao reactivates latent human immunodeficiency virus type 1 provirus. Biochem Biophys Res Commun. 2015; 459(2): 288–93. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWang C, Yang S, Lu H, et al.: A Natural Product from Polygonum cuspidatum Sieb. Et Zucc. Promotes Tat-Dependent HIV Latency Reversal through Triggering P-TEFb's Release from 7SK snRNP. PLoS One. 2015; 10(11): e0142739. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBeans EJ, Fournogerakis D, Gauntlett C, et al.: Highly potent, synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo. Proc Natl Acad Sci U S A. 2013; 110(29): 11698–703. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeChristopher BA, Loy BA, Marsden MD, et al.: Designed, synthetically accessible bryostatin analogues potently induce activation of latent HIV reservoirs in vitro. Nat Chem. 2012; 4(9): 705–10. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWender PA, Nakagawa Y, Near KE, et al.: Computer-guided design, synthesis, and protein kinase C affinity of a new salicylate-based class of bryostatin analogs. Org Lett. 2014; 16(19): 5136–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPandeló José D, Bartholomeeusen K, da Cunha RD, et al.: Reactivation of latent HIV-1 by new semi-synthetic ingenol esters. Virology. 2014; 462-463: 328–39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJiang G, Mendes EA, Kaiser P, et al.: Reactivation of HIV latency by a newly modified Ingenol derivative via protein kinase Cδ-NF-κB signaling. AIDS. 2014; 28(11): 1555–66. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAbreu CM, Price SL, Shirk EN, et al.: Dual role of novel ingenol derivatives from Euphorbia tirucalli in HIV replication: inhibition of de novo infection and activation of viral LTR. PLoS One. 2014; 9(5): e97257. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nJiang G, Mendes EA, Kaiser P, et al.: Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation. PLoS Pathog. 2015; 11(7): e1005066. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDarcis G, Kula A, Bouchat S, et al.: An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression. PLoS Pathog. 2015; 11(7): e1005063. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaird GM, Bullen CK, Rosenbloom DI, et al.: Ex vivo analysis identifies effective HIV-1 latency-reversing drug combinations. J Clin Invest. 2015; 125(5): 1901–12. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nScully E, Alter G: NK Cells in HIV Disease. Curr HIV/AIDS Rep. 2016; 13(2): 85–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones RB, O'Connor R, Mueller S, et al.: Histone deacetylase inhibitors impair the elimination of HIV-infected cells by cytotoxic T-lymphocytes. PLoS Pathog. 2014; 10(8): e1004287. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCicin-Sain L, Sylwester AW, Hagen SI, et al.: Cytomegalovirus-specific T cell immunity is maintained in immunosenescent rhesus macaques. J Immunol. 2011; 187(4): 1722–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHansen SG, Ford JC, Lewis MS, et al.: Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine. Nature. 2011; 473(7348): 523–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHansen SG, Sacha JB, Hughes CM, et al.: Cytomegalovirus vectors violate CD8+ T cell epitope recognition paradigms. Science. 2013; 340(6135): 1237874. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHalper-Stromberg A, Nussenzweig MC: Towards HIV-1 remission: potential roles for broadly neutralizing antibodies. J Clin Invest. 2016; 126(2): 415–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChun TW, Murray D, Justement JS, et al.: Broadly neutralizing antibodies suppress HIV in the persistent viral reservoir. Proc Natl Acad Sci U S A. 2014; 111(36): 13151–6. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHalper-Stromberg A, Lu CL, Klein F, et al.: Broadly neutralizing antibodies and viral inducers decrease rebound from HIV-1 latent reservoirs in humanized mice. Cell. 2014; 158(5): 989–99. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBruel T, Guivel-Benhassine F, Amraoui S, et al.: Elimination of HIV-1-infected cells by broadly neutralizing antibodies. Nat Commun. 2016; 7: 10844. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGrupp SA, Kalos M, Barrett D, et al.: Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013; 368(16): 1509–18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaude SL, Frey N, Shaw PA, et al.: Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med. 2014; 371(16): 1507–17. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGavegnano C, Detorio M, Montero C, et al.: Ruxolitinib and tofacitinib are potent and selective inhibitors of HIV-1 replication and virus reactivation in vitro. Antimicrob Agents Chemother. 2014; 58(4): 1977–86. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHaile WB, Gavegnano C, Tao S, et al.: The Janus kinase inhibitor ruxolitinib reduces HIV replication in human macrophages and ameliorates HIV encephalitis in a murine model. Neurobiol Dis. 2016; pii: S0969-9961(16)30028-6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nStarzl TE, Weil R 3rd, Iwatsuki S, et al.: The use of cyclosporin A and prednisone in cadaver kidney transplantation. Surg Gynecol Obstet. 1980; 151(1): 17–26. PubMed Abstract | Free Full Text\n\nBunjes D, Hardt C, Röllinghoff M, et al.: Cyclosporin A mediates immunosuppression of primary cytotoxic T cell responses by impairing the release of interleukin 1 and interleukin 2. Eur J Immunol. 1981; 11(8): 657–61. PubMed Abstract | Publisher Full Text\n\nAndrieu JM, Even P, Venet A, et al.: Effects of cyclosporin on T-cell subsets in human immunodeficiency virus disease. Clin Immunol Immunopathol. 1988; 47(2): 181–98. PubMed Abstract | Publisher Full Text\n\nMarkowitz M, Vaida F, Hare CB, et al.: The virologic and immunologic effects of cyclosporine as an adjunct to antiretroviral therapy in patients treated during acute and early HIV-1 infection. J Infect Dis. 2010; 201(9): 1298–302. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRizzardi GP, Harari A, Capiluppi B, et al.: Treatment of primary HIV-1 infection with cyclosporin A coupled with highly active antiretroviral therapy. J Clin Invest. 2002; 109(5): 681–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMousseau G, Kessing CF, Fromentin R, et al.: The Tat Inhibitor Didehydro-Cortistatin A Prevents HIV-1 Reactivation from Latency. MBio. 2015; 6(4): e00465. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMediouni S, Jablonski J, Paris JJ, et al.: Didehydro-cortistatin A inhibits HIV-1 Tat mediated neuroinflammation and prevents potentiation of cocaine reward in Tat transgenic mice. Curr HIV Res. 2015; 13(1): 64–79. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHütter G, Nowak D, Mossner M, et al.: Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N Engl J Med. 2009; 360(7): 692–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nTebas P, Stein D, Tang WW, et al.: Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med. 2014; 370(10): 901–10. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nYe L, Wang J, Beyer AI, et al.: Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection. Proc Natl Acad Sci U S A. 2014; 111(26): 9591–6. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWang W, Ye C, Liu J, et al.: CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection. PLoS One. 2014; 9(12): e115987. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSchumann K, Lin S, Boyer E, et al.: Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci U S A. 2015; 112(33): 10437–42. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHou P, Chen S, Wang S, et al.: Genome editing of CXCR4 by CRISPR/cas9 confers cells resistant to HIV-1 infection. Sci Rep. 2015; 5: 15577. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nChan E, Towers GJ, Qasim W: Gene therapy strategies to exploit TRIM derived restriction factors against HIV-1. Viruses. 2014; 6(1): 243–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarthagena L, Parise MC, Ringeard M, et al.: Implication of TRIM alpha and TRIMCyp in interferon-induced anti-retroviral restriction activities. Retrovirology. 2008; 5: 59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeagu MR, Ziegler P, Pertel T, et al.: Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components. J Clin Invest. 2009; 119(10): 3035–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWalker JE, Chen RX, McGee J, et al.: Generation of an HIV-1-resistant immune system with CD34+ hematopoietic stem cells transduced with a triple-combination anti-HIV lentiviral vector. J Virol. 2012; 86(10): 5719–29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWright AV, Nuñez JK, Doudna JA: Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering. Cell. 2016; 164(1–2): 29–44. PubMed Abstract | Publisher Full Text\n\nEbina H, Misawa N, Kanemura Y, et al.: Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep. 2013; 3: 2510. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu W, Kaminski R, Yang F, et al.: RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc Natl Acad Sci U S A. 2014; 111(31): 11461–6. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWang G, Zhao N, Berkhout B, et al.: CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape. Mol Ther. 2016; 24(3): 522–6. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nZhang Y, Yin C, Zhang T, et al.: CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs. Sci Rep. 2015; 5: 16277. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSaayman SM, Lazar DC, Scott TA, et al.: Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex. Mol Ther. 2016; 24(3): 488–98. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLimsirichai P, Gaj T, Schaffer DV: CRISPR-mediated Activation of Latent HIV-1 Expression. Mol Ther. 2016; 24(3): 499–507. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMassanella M, Richman DD: Measuring the latent reservoir in vivo. J Clin Invest. 2016; 126(2): 464–72. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "14023",
"date": "26 May 2016",
"name": "Greg Towers",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14024",
"date": "26 May 2016",
"name": "Nathaniel Landau",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14025",
"date": "26 May 2016",
"name": "Thomas J. Hope",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1009
|
https://f1000research.com/articles/5-1007/v1
|
26 May 16
|
{
"type": "Review",
"title": "Members of the genus Burkholderia: good and bad guys",
"authors": [
"Leo Eberl",
"Peter Vandamme",
"Peter Vandamme"
],
"abstract": "In the 1990s several biocontrol agents on that contained Burkholderia strains were registered by the United States Environmental Protection Agency (EPA). After risk assessment these products were withdrawn from the market and a moratorium was placed on the registration of Burkholderia-containing products, as these strains may pose a risk to human health. However, over the past few years the number of novel Burkholderia species that exhibit plant-beneficial properties and are normally not isolated from infected patients has increased tremendously. In this commentary we wish to summarize recent efforts that aim at discerning pathogenic from beneficial Burkholderia strains.",
"keywords": [
"When the genus Burkholderia was defined in 1992 by Yabuuchi et al. to accommodate most of the former rRNA group II pseudomonads",
"it consisted of only seven species1. Two of these species (Burkholderia pseudomallei and Burkholderia mallei) are primary pathogens of animals and humans",
"two species (Burkholderia caryophylli and Burkholderia gladioli) are known as plant pathogens",
"two species (Burkholderia solanacearum [a plant pathogen] and Burkholderia pickettii [an opportunistic human pathogen]) were later transferred to the genus Ralstonia",
"and the remaining species",
"Burkholderia cepacia",
"was originally described as the causative agent of bacterial rot of onion bulbs2. Since the first description of the genus",
"the number of validly named species has increased to almost one hundred (http://www.bacterio.net/burkholderia.html). During this time",
"it has become apparent that this genus has tremendous biotechnological potential",
"with species that produce a large variety of commercially important hydrolytic enzymes and bioactive substances",
"that promote plant growth and health",
"and that can degrade various recalcitrant pollutants. Yet their agricultural and industrial use is severely limited due to the potential threat that some strains pose to human health3. In addition to B. pseudomallei and B. mallei",
"it is a group of currently 20 closely related bacterial species in particular",
"referred to as the Burkholderia cepacia complex (Bcc)",
"which have emerged as opportunistic pathogens that can cause severe infections in cystic fibrosis (CF) and immunocompromised patients4–6. However",
"virtually all Bcc species have also been isolated from the natural environment",
"often from soil samples or from the rhizosphere of various plants. The use of Burkholderia in agricultural applications is therefore considered a double-edged sword",
"and a lot of effort has been invested into discriminating between the beneficial environmental (the good) and the clinical (the bad) Burkholderia strains7",
"8. Recently",
"these efforts have gained momentum",
"as many new Burkholderia species have been identified in environmental samples that exhibit potentially valuable beneficial traits. These species are believed to be safe for applications",
"as there are very rarely clinical reports that they would pose a risk to human health."
],
"content": "The genus Burkholderia: past and present\n\nWhen the genus Burkholderia was defined in 1992 by Yabuuchi et al. to accommodate most of the former rRNA group II pseudomonads, it consisted of only seven species1. Two of these species (Burkholderia pseudomallei and Burkholderia mallei) are primary pathogens of animals and humans, two species (Burkholderia caryophylli and Burkholderia gladioli) are known as plant pathogens, two species (Burkholderia solanacearum [a plant pathogen] and Burkholderia pickettii [an opportunistic human pathogen]) were later transferred to the genus Ralstonia, and the remaining species, Burkholderia cepacia, was originally described as the causative agent of bacterial rot of onion bulbs2. Since the first description of the genus, the number of validly named species has increased to almost one hundred (http://www.bacterio.net/burkholderia.html). During this time, it has become apparent that this genus has tremendous biotechnological potential, with species that produce a large variety of commercially important hydrolytic enzymes and bioactive substances, that promote plant growth and health, and that can degrade various recalcitrant pollutants. Yet their agricultural and industrial use is severely limited due to the potential threat that some strains pose to human health3. In addition to B. pseudomallei and B. mallei, it is a group of currently 20 closely related bacterial species in particular, referred to as the Burkholderia cepacia complex (Bcc), which have emerged as opportunistic pathogens that can cause severe infections in cystic fibrosis (CF) and immunocompromised patients4–6. However, virtually all Bcc species have also been isolated from the natural environment, often from soil samples or from the rhizosphere of various plants. The use of Burkholderia in agricultural applications is therefore considered a double-edged sword, and a lot of effort has been invested into discriminating between the beneficial environmental (the good) and the clinical (the bad) Burkholderia strains7,8. Recently, these efforts have gained momentum, as many new Burkholderia species have been identified in environmental samples that exhibit potentially valuable beneficial traits. These species are believed to be safe for applications, as there are very rarely clinical reports that they would pose a risk to human health.\n\n\nBurkholderia species in the environment\n\nRecent work has shown that members of the genus Burkholderia are common soil inhabitants and that their biogeographic distribution is strongly affected by soil pH9–12. Due to their intrinsic acid tolerance, Burkholderia strains have a competitive advantage in acidic soils but are outcompeted in alkaline soils. Moreover, it has been reported that Burkholderia significantly co-occurs with a wide range of fungi, which normally also prefer acidic environments13. This finding is in line with reports demonstrating that many Burkholderia species can form either antagonistic or mutualistic interactions with fungi. While antagonistic behavior of Burkholderia species is well described and is dependent on the production of a large variety of antifungal compounds (for a review, see 14), other species have been demonstrated to live in mutualistic associations with fungi. A well-investigated example is represented by the association of Burkholderia terrae and Lyophyllum species, for which it was shown that the bacteria can not only use the hyphae of the fungus for transportation and dispersal but also use fungal exudates as nutrients15–17. This is in full agreement with the finding that Burkholderia strains are among the main consumers of carbon released from arbuscular mycorrhizal fungi18. Another intriguing example is Burkholderia rhizoxinica, which invades hyphae of the fungus Rhizopus microsporus19,20, the causative agent of rice seedling blight. The symbiont is involved in the biosynthesis of the antimitotic toxin rhizoxin21, which efficiently stalls plant cell division. In the absence of the endosymbiont, the fungus was found to be unable to reproduce vegetatively22.\n\nAnother emerging theme is the tight association of some Burkholderia species with plants. Over the past few years, the number of novel plant-associated Burkholderia species has increased tremendously. These new species show various degrees of plant dependence, with some strains living freely in the rhizosphere, exhibiting an endophytic lifestyle, nodulating legumes, or, most intriguingly, forming an obligate leaf symbiosis with their host plants. Burkholderia species have been frequently isolated from diverse surface-sterilized plants (e.g. 23–27). Probably the best studied endophytic Burkholderia strain is Burkholderia phytofirmans PsJN, which was originally isolated from onion roots and was subsequently demonstrated to establish endophytic populations in various plants28,29. Interestingly, B. phytofirmans is not only capable of protecting plants from pathogens (through an unknown mechanism) but was also shown to increase the plants’ stress resistance, particularly against low temperatures, high salt, and drought30–32. Some Burkholderia species have been shown to be specifically associated with Sphagnum mosses33,34. Since Moulin et al. demonstrated that two Burkholderia species, which were isolated from root nodules of a legume, possessed nodulation genes35, many more nodulating Burkholderia species have been described (for recent studies, see 36–38). Although these strains have mainly been isolated from Mimosa species, recent work showed that some Burkholderia strains can also nodulate fynbos legumes in South Africa39–43. Some plant genera of the Rubiaceae and Primulaceae families carry members of the genus Burkholderia within leaf nodules44–47. This unique association is the only known example of an obligate plant-bacterium symbiosis with both partners being unable to exist outside the symbiotic association. The bacterial symbiont is thought to be hereditarily transmitted to the progeny via colonization of the developing seeds. Although the molecular nature of the leaf nodule symbiosis is still unknown, it was recently shown that the bacterial symbiont produces large amounts of secondary metabolites, which appear to protect the plants from herbivores48,49.\n\nFinally, a large body of evidence demonstrates that many insect species harbor symbiotic bacteria of the genus Burkholderia50–53. The association of Burkholderia species with the bean bug Riptortus pedestris has emerged as a promising experimental model to study the molecular mechanisms involved in insect-bacterium symbiosis54,55. This symbiosis appears to be particularly tight, as it was recently reported that the insect has a previously unrecognized animal organ used to specifically sort the symbiont into the posterior gut region, which is devoid of food flow and is transformed into an isolated organ for symbiosis56.\n\nWe are convinced that these examples represent just the tip of the iceberg and that many more Burkholderia fungus/plant/insect associations will be discovered in the future.\n\n\nCan we tell the good from the bad by taxonomy?\n\nPhylogenetic investigations have provided evidence that members of the genus Burkholderia can be divided into two main lineages (Figure 1) and several species that represent unique lines of descent. One clade comprises pathogens of humans, animals, and plants, including B. pseudomallei, B. mallei, and Burkholderia glumae, as well as the Bcc species. However, this clade also contains many strains that can be used for plant growth promotion and biocontrol of plant pests, including Burkholderia vietnamiensis TVV74 and Burkholderia ambifaria AMMD, respectively57. Ironically, although Burkholderia cenocepacia is generally considered the most problematic Bcc species in patients with CF58, recently a genome sequence of a plant-beneficial endophytic B. cenocepacia strain with both biocontrol and plant-growth-promoting characteristics was reported59. Also, non-Bcc Burkholderia species within this clade can have both beneficial and harmful properties. One intriguing case with great potential for agricultural applications is represented by Burkholderia gladioli, which is a well-known pathogen of plants (e.g. causing rice panicle blight)60 as well as humans61–63. However, recent work has demonstrated that some B. gladioli strains live endophytically within various wild and ancient Zea plants without causing any disease symptoms64,65. In contrast, this endophyte was shown to produce an unidentified antifungal compound in planta and was able to suppress the fungal pathogen Sclerotinia homoeocarpa66.\n\nThe second main phylogenetic Burkholderia cluster contains many plant-beneficial environmental Burkholderia species, as mentioned above67. Several of these species have been reported to fix nitrogen, to be capable of nodulating legumes, to promote plant growth, and to degrade recalcitrant compounds68. Given that species of this cluster are only rarely isolated from infected patients69–71, they are often considered to pose no risk to human health and have therefore been suggested to be promising candidates for applications in biocontrol, biofertilization, and bioremediation72–74. In our opinion, this is a wishful, potentially dangerous, and certainly oversimplified view.\n\nThe alignment was performed using SINA v1.2.11 (http://www.arb-silva.de/aligner/)93. After gap removal with TrimAl94, the final alignment consisted of 1289 positions. The phylogenetic reconstruction was conducted with MEGA695 using Tamura-Nei evolutionary model96 with gamma rate distribution (five gamma categories and 70% of invariable sites). Bootstrap test values are shown if greater than 50%. Some phenotypic characteristics are indicated. No boxes indicate that no information is available. The information on the presence of the type III secretion system is taken from 73.\n\nDividing the genus Burkholderia into two genera was recently proposed, with the novel genus Paraburkholderia containing the primarily environmental species (the alleged good ones) to demarcate them from Burkholderia sensu stricto, which comprises environmental, human clinical, and phytopathogenic species (the alleged bad ones)72. In this study, the percentage guanine plus cytosine content and conserved indels in whole genome sequences of some 25 formally named Burkholderia species and several unclassified strains were studied. Species belonging to the Burkholderia sensu stricto clade were characterized by a percentage guanine plus cytosine content of 65 to 69% and shared six conserved sequence indels, while all other Burkholderia strains examined had a percentage guanine plus cytosine content of 61 to 65% and shared two conserved sequence indels. The phylogenetic heterogeneity among the remaining Burkholderia species as revealed by 16S rRNA-based divergence and by differences in the distribution of 22 additional conserved sequence indels was ignored, as the authors proposed reclassifying all remaining Burkholderia species into a single novel genus, Paraburkholderia72. These novel names were subsequently validated75 and now have formal standing in bacterial nomenclature. The scientific community may adopt these novel names or not. Authors who are convinced that these name changes are ill founded can continue to work with the original species names, as all these were validly published.\n\nA recent study employed comparative genomics to assess the pathogenic potential of environmental strains on the basis of the presence or absence of known virulence factors73. This bioinformatic study clearly showed that many virulence factors, including the type III, IV, and VI secretion systems, are mostly found in representatives of the Burkholderia sensu stricto clade while they are often absent in strains of the Paraburkholderia clade. The authors also show that Paraburkholderia strains exhibit no virulence in a Caenorhabditis elegans infection model. While these are valuable approaches, they also have their caveats. Many virulence factors of Burkholderia species have been shown to be host specific, and there is little correlation between the different infection models commonly used, e.g. C. elegans, Galleria mellonella, and Drosophila melanogaster. This probably reflects the need for Burkholderia strains to compete for survival in diverse habitats such as soil, plants, insects, and mammalian hosts. Only very few universal virulence factors could be identified in B. cenocepacia (namely quorum sensing, siderophore production, and lipopolysaccharide biosynthesis) and therefore extrapolations from non-mammalian infection models to mammalian infections, particularly to chronic CF lung infections, must be made with caution76,77. For example, most Burkholderia multivorans strains show no virulence in a C. elegans or G. mellonella infection model78,79, although most virulence factors that were suggested to be indicative for pathogenic Burkholderia species could be identified in this Bcc species73. Yet B. multivorans (along with B. cenocepacia) is one of the predominant Burkholderia species infecting people with CF58,80. On the other hand, B. cenocepacia strain H11181, which is closely related to strains of the epidemic ET12 lineage (e.g. J2315 and K56-2), did not cause acute symptoms in the infected CF patient from whom it was isolated and was cleared after a 6-month co-infection period with Pseudomonas aeruginosa82, while infections with strains of the ET12 lineage have resulted in high mortality among patients58,83. In contrast to its clinical impact, strain H111 shows a similar level of pathogenicity in the G. mellonella and C. elegans infection models to K56-2 (an ET12 lineage strain) and both strains are much more virulent in these models than J2315 (another ET12 lineage strain)77.\n\nThese examples strongly suggest that neither the presence of virulence genes in a strain nor acute virulence as assessed in routinely used non-mammalian infection models is an absolutely reliable predictor of clinical prevalence or outcome in CF patients. The taxonomic position of a strain is also not an unambiguous indicator for its pathogenic potential and thus decisions on the industrial or biotechnological use of a Burkholderia strain can be made only on a case-by-case basis after careful molecular and phenotypic characterization of the strain. On the comparative genomics side, it will be interesting to see whether the co-occurrence of certain genes may be a suitable indicator of the phenotypic potential of a strain, as has recently been proposed in the case of plant-growth-promoting bacteria84.\n\n\nThe use of Burkholderia as biocontrol agents\n\nAlthough endophytic or nitrogen-fixing Burkholderia strains show great promise as agents for plant growth promotion and bioremediation, it should be kept in mind that in terms of biocontrol applications the most outstanding property of Burkholderia strains is the production of various compounds with potent antifungal activity14,85. In fact, several Bcc strains have been registered by the United States Environmental Protection Agency (EPA) for use as biocontrol agents against phytopathogenic fungi, including Deny®, Blue Circle®, and Intercept®, in the 1990s. However, after risk assessment, these products were withdrawn from the market and the EPA placed a moratorium on the registration of products containing Bcc species (https://www.gpo.gov/fdsys/pkg/FR-2004-09-29/pdf/04-21695.pdf). Would it be possible to replace these Bcc-based biocontrol agents with strains of the Paraburkholderia lineage? Literature research, genome mining, and experimental evidence (Figure 1) have revealed that only three species of the Paraburkholderia cluster, namely Burkholderia phenazinium, Burkholderia megapolitana, and Burkholderia bryophila, all of which have been isolated from mosses86, show antifungal activity. In contrast, most strains of the Bcc and many of the human and plant pathogenic species produce antifungal compounds85. Given that most antifungal agents exhibit more general toxic effects in eukaryotic organisms, these compounds may contribute to the virulence of a strain. B. phenazinium was reported to produce the phenazine iodinin87, which exhibits not only high anti-microbial but also cytotoxic activity. While iodinin may be valuable for clinical purposes, as it is potent against leukemia cell lines88, it may not be useful for biocontrol applications. To our knowledge, the antifungal compounds produced by B. megapolitana and B. bryophila have not been identified nor has their pathogenic potential been evaluated in an infection model. In conclusion, while many Bcc strains have been demonstrated to exhibit excellent biocontrol activities, there are only very few Paraburkholderia strains that are potentially useful for biocontrol purposes.\n\n\nIs there a safe Burkholderia strain?\n\nGiven the lack of reported cases in the literature, many strains of the Paraburkholderia lineage seem unlikely to cause infections in humans and therefore could be considered for agricultural applications. The same may also apply to some strains of the Burkholderia lineage, as has recently been suggested for the Bcc strain Burkholderia contaminans MS14, which was found to possess multiple antimicrobial biosynthesis genes but not major genetic loci required for pathogenesis89. While the phylogenetic status of a strain may be helpful as a first approximation of the pathogenic potential of a strain, it is clear that the Paraburkholderia lineage contains some pathogenic strains and that several Bcc strains exhibit good biocontrol properties and attenuated virulence. Hence, independent of a strain’s phylogenetic status, a thorough characterization of a strain will be required before it can be considered safe. It will be important to use well-established infection models such as the mouse model90 for the assessment of the potential pathogenicity of a strain and to carefully examine whether related strains have been isolated from infected humans. Likewise, the biocontrol activity of the strain has to be tested in field trials. It is also worth noting that several species of the Paraburkholderia clade, including the well-investigated endophyte B. phytofirmans, are unable to grow at 37°C (in contrast to Burkholderia sensu stricto species), a property that is considered to be essential to infect and colonize humans. The capability to grow at 37°C has recently been proposed as a simple means to differentiate between pathogenic and non-pathogenic Stenotrophomonas maltophilia and Stenotrophomonas rhizophila isolates91. Representatives of the latter species have therefore been suggested to provide an alternative to biotechnological applications without posing any risk to human health92. An important line of future research will therefore be to assess the pathogenicity of environmental strains in suitable infection models, particularly using a mammalian host at 37°C, and ideally in multispecies infection scenarios, which may more accurately reflect the genuine clinical situation.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nFinancial support from the Swiss National Fund (Project 3100A0-104215) to Leo Eberl is gratefully acknowledged.\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 are grateful to Marta Pinto for generating the phylogenetic tree shown in Figure 1. We would like to thank all present and past members of our working groups for their various contributions.\n\n\nReferences\n\nYabuuchi E, Kosako Y, Oyaizu H, et al.: Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol Immunol. 1992; 36(12): 1251–75. PubMed Abstract | Publisher Full Text\n\nBurkholder WH: Sour skin, a bacterial rot of Onion bulbs. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nHo YN, Huang CC: Draft Genome Sequence of Burkholderia cenocepacia Strain 869T2, a Plant-Beneficial Endophytic Bacterium. Genome Announc. 2015; 3(6): pii: e01327-15. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nNaughton LM, An SQ, Hwang I, et al.: Functional and genomic insights into the pathogenesis of Burkholderia species to rice. Environ Microbiol. 2016; 18(3): 780–90. PubMed Abstract | Publisher Full Text\n\nZhou F, Ning H, Chen F, et al.: Burkholderia gladioli infection isolated from the blood cultures of newborns in the neonatal intensive care unit. Eur J Clin Microbiol Infect Dis. 2015; 34(8): 1533–7. PubMed Abstract | Publisher Full Text\n\nImataki O, Kita N, Nakayama-Imaohji H, et al.: Bronchiolitis and bacteraemia caused by Burkholderia gladioli in a non-lung transplantation patient. New Microbes New Infect. 2014; 2(6): 175–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSegonds C, Clavel-Batut P, Thouverez M, et al.: Microbiological and epidemiological features of clinical respiratory isolates of Burkholderia gladioli. J Clin Microbiol. 2009; 47(5): 1510–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnston-Monje D, Raizada MN: Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology. PLoS One. 2011; 6(6): e20396. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEttinger CL, Shehata HR, Johnston-Monje D, et al.: Draft Genome Sequence of Burkholderia gladioli Strain UCD-UG_CHAPALOTE (Phylum Proteobacteria). Genome Announc. 2015; 3(1): pii: e01462-14. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nShehata HR, Lyons EM, Jordan KS, et al.: Bacterial endophytes from wild and ancient maize are able to suppress the fungal pathogen Sclerotinia homoeocarpa. J Appl Microbiol. 2016; 120(3): 756–69. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSuárez-Moreno ZR, Caballero-Mellado J, Coutinho BG, et al.: Common features of environmental and potentially beneficial plant-associated Burkholderia. Microb Ecol. 2012; 63(2): 249–66. PubMed Abstract | Publisher Full Text\n\nPérez-Pantoja D, Nikel PI, Chavarría M, et al.: Endogenous stress caused by faulty oxidation reactions fosters evolution of 2,4-dinitrotoluene-degrading bacteria. PLoS Genet. 2013; 9(8): e1003764. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCoenye T, Goris J, Spilker T, et al.: Characterization of unusual bacteria isolated from respiratory secretions of cystic fibrosis patients and description of Inquilinus limosus gen. nov., sp. nov. J Clin Microbiol. 2002; 40(6): 2062–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGerrits GP, Klaassen C, Coenye T, et al.: Burkholderia fungorum septicemia. Emerg Infect Dis. 2005; 11(7): 1115–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeris ZZ, Van Rostenberghe H, Habsah H, et al.: First isolation of Burkholderia tropica from a neonatal patient successfully treated with imipenem. Int J Infect Dis. 2010; 14(1): e73–4. PubMed Abstract | Publisher Full Text\n\nSawana A, Adeolu M, Gupta RS: Molecular signatures and phylogenomic analysis of the genus Burkholderia: proposal for division of this genus into the emended genus Burkholderia containing pathogenic organisms and a new genus Paraburkholderia gen. nov. harboring environmental species. Front Genet. 2014; 5: 429. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAngus AA, Agapakis CM, Fong S, et al.: Plant-associated symbiotic Burkholderia species lack hallmark strategies required in mammalian pathogenesis. PLoS One. 2014; 9(1): e83779. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nEstrada-de los Santos P, Vinuesa P, Martínez-Aguilar L, et al.: Phylogenetic analysis of burkholderia species by multilocus sequence analysis. Curr Microbiol. 2013; 67(1): 51–60. PubMed Abstract | Publisher Full Text\n\nOren A, Garrity GM: List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2015; 65(11): 3763–7. PubMed Abstract | Publisher Full Text\n\nSchwager S, Agnoli K, Köthe M, et al.: Identification of Burkholderia cenocepacia strain H111 virulence factors using nonmammalian infection hosts. Infect Immun. 2013; 81(1): 143–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUehlinger S, Schwager S, Bernier SP, et al.: Identification of specific and universal virulence factors in Burkholderia cenocepacia strains by using multiple infection hosts. Infect Immun. 2009; 77(9): 4102–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCardona ST, Wopperer J, Eberl L, et al.: Diverse pathogenicity of Burkholderia cepacia complex strains in the Caenorhabditis elegans host model. FEMS Microbiol Lett. 2005; 250(1): 97–104. PubMed Abstract | Publisher Full Text\n\nSeed KD, Dennis JJ: Development of Galleria mellonella as an alternative infection model for the Burkholderia cepacia complex. Infect Immun. 2008; 76(3): 1267–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZlosnik JE, Zhou G, Brant R, et al.: Burkholderia species infections in patients with cystic fibrosis in British Columbia, Canada. 30 years' experience. Ann Am Thorac Soc. 2015; 12(1): 70–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nCarlier A, Agnoli K, Pessi G, et al.: Genome Sequence of Burkholderia cenocepacia H111, a Cystic Fibrosis Airway Isolate. Genome Announc. 2014; 2(2): pii: e00298-14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGeisenberger O, Givskov M, Riedel K, et al.: Production of N-acyl-L-homoserine lactones by P. aeruginosa isolates from chronic lung infections associated with cystic fibrosis. FEMS Microbiol Lett. 2000; 184(2): 273–8. PubMed Abstract\n\nDrevinek P, Mahenthiralingam E: Burkholderia cenocepacia in cystic fibrosis: epidemiology and molecular mechanisms of virulence. Clin Microbiol Infect. 2010; 16(7): 821–30. PubMed Abstract | Publisher Full Text\n\nBruto M, Prigent-Combaret C, Muller D, et al.: Analysis of genes contributing to plant-beneficial functions in Plant Growth-Promoting Rhizobacteria and related Proteobacteria. Sci Rep. 2014; 4: 6261. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSchmidt S, Blom JF, Pernthaler J, et al.: Production of the antifungal compound pyrrolnitrin is quorum sensing-regulated in members of the Burkholderia cepacia complex. Environ Microbiol. 2009; 11(6): 1422–37. PubMed Abstract | Publisher Full Text\n\nVandamme P, Opelt K, Knöchel N, et al.: Burkholderia bryophila sp. nov. and Burkholderia megapolitana sp. nov., moss-associated species with antifungal and plant-growth-promoting properties. Int J Syst Evol Microbiol. 2007; 57(Pt 10): 2228–35. PubMed Abstract | Publisher Full Text\n\nTurner JM, Messenger AJ: Occurrence, biochemistry and physiology of phenazine pigment production. Adv Microb Physiol. 1986; 27: 211–75. PubMed Abstract | Publisher Full Text\n\nSletta H, Degnes KF, Herfindal L, et al.: Anti-microbial and cytotoxic 1,6-dihydroxyphenazine-5,10-dioxide (iodinin) produced by Streptosporangium sp. DSM 45942 isolated from the fjord sediment. Appl Microbiol Biotechnol. 2014; 98(2): 603–10. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDeng P, Wang X, Baird SM, et al.: Comparative genome-wide analysis reveals that Burkholderia contaminans MS14 possesses multiple antimicrobial biosynthesis genes but not major genetic loci required for pathogenesis. Microbiologyopen. 2016. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSokol PA, Darling P, Woods DE, et al.: Role of ornibactin biosynthesis in the virulence of Burkholderia cepacia: characterization of pvdA, the gene encoding L-ornithine N5-oxygenase. Infect Immun. 1999; 67(9): 4443–55. PubMed Abstract | Free Full Text\n\nAlavi P, Starcher MR, Thallinger GG, et al.: Stenotrophomonas comparative genomics reveals genes and functions that differentiate beneficial and pathogenic bacteria. BMC Genomics. 2014; 15(1): 482. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBerg G, Martinez JL: Friends or foes: can we make a distinction between beneficial and harmful strains of the Stenotrophomonas maltophilia complex? Front Microbiol. 2015; 6: 241. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPruesse E, Peplies J, Glöckner FO: SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics. 2012; 28(14): 1823–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCapella-Gutiérrez S, Silla-Martínez JM, Gabaldón T: trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009; 25(15): 1972–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTamura K, Stecher G, Peterson D, et al.: MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013; 30(12): 2725–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTamura K, Nei M: Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol. 1993; 10(3): 512–26. PubMed Abstract"
}
|
[
{
"id": "14009",
"date": "26 May 2016",
"name": "Gabriele Berg",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14010",
"date": "26 May 2016",
"name": "Jorge Leitão",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "14012",
"date": "26 May 2016",
"name": "Vittorio Venturi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1007
|
https://f1000research.com/articles/5-999/v1
|
25 May 16
|
{
"type": "Review",
"title": "The germinal center antibody response in health and disease",
"authors": [
"Anthony L. DeFranco"
],
"abstract": "The germinal center response is the delayed but sustained phase of the antibody response that is responsible for producing high-affinity antibodies of the IgG, IgA and/or IgE isotypes. B cells in the germinal center undergo re-iterative cycles of somatic hypermutation of immunoglobulin gene variable regions, clonal expansion, and Darwinian selection for cells expressing higher-affinity antibody variants. Alternatively, selected B cells can terminally differentiate into long-lived plasma cells or into a broad diversity of mutated memory B cells; the former secrete the improved antibodies to fight an infection and to provide continuing protection from re-infection, whereas the latter may jumpstart immune responses to subsequent infections with related but distinct infecting agents. Our understanding of the molecules involved in the germinal center reaction has been informed by studies of human immunodeficiency patients with selective defects in the production of antibodies. Recent studies have begun to reveal how innate immune recognition via Toll-like receptors can enhance the magnitude and selective properties of the germinal center, leading to more effective control of infection by a subset of viruses. Just as early insights into the nature of the germinal center found application in the development of the highly successful conjugate vaccines, more recent insights may find application in the current efforts to develop new generations of vaccines, including vaccines that can induce broadly protective neutralizing antibodies against influenza virus or HIV-1.",
"keywords": [
"germinal center",
"germinal center response",
"somatic hypermutation",
"innate immunity"
],
"content": "Introduction\n\nThe germinal center (GC) reaction is a critical part of the antibody response in which antigen-specific B lymphocytes undergo a sustained period of rapid cellular proliferation, high-level mutagenesis of their antibody genes, and stringent Darwinian selection for those B cells within the GC that make higher-affinity antibodies. The output of the GC is both memory B cells and antibody-secreting cells (called “plasma cells”), and the affinity of both increases for weeks to months, as long as the GC persists, resulting in “affinity maturation” of the antibodies in the blood1. At the same time, there is a change in the type of antibodies being produced, from IgM to IgG, IgA, or IgE, which have more specialized properties with respect to their localization and effector functions.\n\nWhile somatic mutation and antibody class switch occur to some extent outside of GCs, these processes occur to a much greater extent in the GC. Moreover, whereas the plasma cells generated prior to the GC are almost all programmed to die after a few days, many of the plasma cells generated from the GC can migrate to the bone marrow, where they access survival niches that allow them to live for years1,2. These long-lived plasma cells are responsible for more than half of the IgG in blood and provide a continual protection against re-infection with viruses and microbes. IgA and IgE have somewhat distinct biology, but nonetheless these isotypes also may provide a more long-lasting protection than the more rapidly turning over IgM.\n\nThis review will cover recent progress in understanding the cellular and molecular mechanisms controlling the GC response and recent evidence that innate immune pathways can substantially impact the quality of the GC response. In addition, the importance of the GC response in health and disease will be highlighted. As demonstrated by the susceptibilities of immunodeficient individuals with genetic defects in genes required for the GC response, much of immunity from re-infection with pathogens seen previously and of the success of vaccination can be traced to the GC reaction and its output of long-lived plasma cells, memory B cells, and memory helper T cells. Indeed, an understanding of the basics of T cell-dependent antibody responses leading to GCs was used to develop the improved “conjugate vaccines” against a number of bacterial pathogens, starting in the 1990s3. Efforts to improve current vaccines and to create new vaccines against difficult targets also hope to take advantage of the rapidly increasing understanding of the GC reaction. Development of vaccines that induce more broadly crossreactive neutralizing antibodies against influenza virus4,5 and HIV-16 is an especially active effort at this time. Conversely, the pathogenic autoantibodies made in some autoimmune diseases likely emanate from the GC, so approaches to inhibiting the GC response may have therapeutic value in such patients.\n\n\nEarly response to antigen and commitment to the germinal center reaction\n\nNaïve B cells and T cells encounter antigen in separate regions of the secondary lymphoid organ where antigen is first concentrated (spleen, lymph nodes, or Peyer’s patches for antigen in the blood, tissues, and gut lumen, respectively). B cells recognize antigen via the B-cell antigen receptor (BCR), which is a complex between a transmembrane form of the antibody made by that B cell (membrane immunoglobulin), with a heterodimer of two signaling chains called Igα and Igβ. T cells use a similar receptor complex, the T-cell receptor (TCR), containing similar but distinct polypeptides. Rather than recognizing antigen in its native configuration, the TCR recognizes a peptide derived from the antigen bound to the binding groove of one of two types of similar cell surface molecules encoded in the major histocompatibility complex (MHC) and therefore called MHC class I and class II molecules. Antigen encounter by antigen-specific B cells induces changes in expression of receptors for chemotactic cues such as chemokines that direct migration of the cells out of the B cell-rich follicle and to the area between the follicles and the T-cell zone7. Complementary changes are induced in parallel in the antigen-stimulated helper T cells, causing them to migrate to the same place as the antigen-stimulated B cells8. Thus, initial antigen encounter causes antigen-stimulated B cells and helper T cells to relocalize to the same zone within the lymphoid organ and thereby facilitate their interaction (Figure 1).\n\nAntigen-specific B cells contact the incoming antigen (red polygon) in the B cell-rich follicles of secondary lymphoid tissues. Low levels of signaling from the B-cell antigen receptor (BCR) are sufficient to alter responsiveness to chemotactic stimuli such as chemokines, causing the B cell to move out of the follicle into the extrafollicular region adjacent to the T-cell zone. Low-level BCR signaling is also sufficient to promote internalization of the BCR-antigen complex into endocytic compartments, where protein antigens are degraded into peptides (red triangles), which are loaded onto major histocompatibility complex (MHC) class II molecules. Once bound with a peptide, MHC class II molecules traffic to the cell surface, where they can be recognized by the T-cell receptors (TCRs) of T cells that are also specific for the same antigen. Naïve T cells are found in the T-cell zone, where they may become activated by dendritic cells presenting antigenic peptides bound to class II MHC molecules. This activation induces movement of the helper T cell to the boundary of the T-cell zone and the follicle, where they scan B cells for the presence of their TCR ligand. TCR recognition induces prolonged contact with the antigen-presenting B cell and expression of CD40L and of cytokines, which promote activation and clonal expansion of antigen-specific B cells. Interactions between B cells and helper T cells occur approximately 24 hours after antigen arrival in the secondary lymphoid organ and continue in this location until the start of the GC reaction, on day 4 or 5.\n\nB cells also use their BCR to internalize antigen, which is critical for their interactions with helper T cells. The internalized antigen becomes partially degraded in endocytic vesicles and, if it has an attached protein component, the resulting antigen-derived peptides are loaded onto MHC class II molecules, which then traffic to the B-cell surface for recognition by antigen-specific helper T cells9. Recognition by the activated helper T cell of its specific peptide/MHC II complex on the B cell focuses the former’s stimulatory signals on B cells that can recognize an element of the same antigen complex (Figure 1).\n\nThe phase of an antibody response that occurs prior to the GC is referred to as the extrafollicular response. This early phase of the response is characterized by rapid clonal expansion of the antigen-specific B cells and helper T cells, and their interactions contribute importantly to each other’s clonal expansion. During the pre-GC phase, antigen-specific B cells and helper T cells make fate choices between becoming effector cells (plasma cells or effector CD4 T cells) versus migrating into the nascent GC and participating in the slower but higher-quality GC antibody response1. After the first day or so of the response, the helper T cells that have made contact with antigen-specific B cells are recognizable as distinct from effector CD4 T cells and are already referred to as follicular helper T (TFH) cells, although they do not actually enter the B cell-rich follicles until the GC response proper is ready to start10. By 4–5 days after antigen encounter, some antigen-specific B cells have terminally differentiated into plasma cells, some have become early memory B cells, and some have upregulated the transcriptional repressor Bcl6 and thereby have committed to the GC response11. The GC-committed cells migrate back into the follicle where they first encountered antigen. Although how cells choose to become memory B cells is poorly understood, it is clear that the choice between GC phenotype and plasma cell phenotype is determined by the competing cellular programs driven by Bcl6 (GC program) and the transcriptional regulator BLIMP-1 (plasma cell program)12. Remarkably, the same two transcriptional regulators drive the corresponding programs in CD4 T cells: BLIMP-1 drives the program of effector CD4 T cells, and Bcl6 drives the TFH cell program, both during the pre-GC phase of the response and also during the GC phase proper10. The effector versus TFH cell fate decision likely occurs soon after antigen encounter and prior to migration to meet up with antigen-stimulated B cells; nonetheless, full attainment of the TFH cell program, including the ability to localize to the GC itself, requires reinforcement by interaction with antigen-presenting B cells in the extrafollicular region.\n\nThe GC is first evident histologically at about day 5 of the immune response, and at this point both GC B cells and TFH cells take on characteristic cell surface phenotypes that allow their enumeration and isolation. GC B cells gain expression of Fas, PD-L1, and carbohydrates detected with the lectin peanut agglutinin and with the monoclonal antibody GL-7 and also downregulate IgD. TFH cells further upregulate CXCR5 and PD-1, such that the cells with highest expression of these markers are those CD4 T cells found in the GC, whereas CD4 T cells with somewhat lower levels of these proteins are found in the T-B boundary where interactions with antigen-specific B cells initiate10.\n\n\nMechanism of class switch and somatic mutation\n\nAs mentioned above, central to the GC pathway are hypermutation of Ig gene variable regions and class-switch recombination to change the constant regions of the Ig heavy chain. Strongly upregulated in GC B cells is the gene encoding activation-induced cytidine deaminase (AID), an enzyme that converts cytidine in DNA to uridine. In this way, AID initiates somatic hypermutation and class-switch recombination13. AID is expressed at a low level in B cells during the pre-GC phase of the antibody response and is further upregulated in GC B cells, which corresponds to the relative rates of mutation and class switch in these two phases of the antibody response. People and mice with deleterious mutations of the gene encoding AID exhibit strong defects in both somatic mutation and class-switch recombination and correspondingly poor antibody-mediated immunity14,15. It was initially surprising that the same enzyme was required for both of these events, leading to the hypothesis that the role of AID was indirect15. More recently, however, a strong consensus has emerged that AID is directly responsible both for somatic hypermutation and for class-switch recombination. The enzymology of DNA repair is complex, but the mutagenic effect of AID is enhanced by error-prone DNA repair mechanisms, which can spread mutation to bases near the originally targeted cytidine residue13. Thus, the properties of somatic hypermutation are readily accounted for by direct action of AID combined with various DNA repair mechanisms. Class-switch recombination starts with double-stranded DNA breaks in the “switch” regions adjacent to the exons encoding the various Ig heavy-chain constant regions. Switch regions have a very high density of AID-preferred sequence motifs16, so it is likely that AID deaminates multiple nearby cytidines on both DNA strands, followed by a double-stranded DNA break, which can lead to class-switch recombination17,18. Although AID can cause mutations in transcribed genes other than Ig genes, its action appears to be concentrated in switch regions and in the variable regions of assembled antibody genes. How this occurs remains an unsolved problem.\n\n\nRepeated cycles of somatic hypermutation, proliferation, and selection\n\nThe GC reaction first becomes evident after a substantial clonal expansions of the few clonally distinct antigen-specific B cells that are the founders of each GC. Intravital microscopic imagining studies have shown that GC B cells are highly restricted to their GC and do not travel between different GCs in the same lymph node, spleen, or Peyer’s patch or do so infrequently19,20. This restriction of GC B cells and their clonal progeny to a single GC is important because around day 7, there begins a stringent affinity-based selection within a GC in which the mutated expanded cells within a given GC compete with one another on the basis of their affinities for antigen. Thus, as an immune response has several GCs, each seeded with different B cells at the start of the GC phase of the response, affinity maturation can proceed independently in each GC, likely maintaining a diversity of epitopes recognized by the different GC B cells. It should be noted, however, that entry into an active GC by naïve or recently activated B cells readily occurs11, so it is possible for new competitors to enter a GC after it has started its selective processes and impact the Darwinian selection in the GC. Biologically, antibodies to some epitopes are likely to be more protective than antibodies to other epitopes; for example, antibodies that block virus infection of cells (“neutralization”) typically bind to regions on the virus particle that dock with the target cell. Thus, an important consideration in vaccine design is whether the induced GC response is adequately focused on the desired epitopes21.\n\nAt about the same time that affinity selection begins within the GC, the GC can be seen in histological sections to have a “dark” zone, which contains many dividing B cells, and a “light” zone, which contains GC B cells as well as TFH cells and antigen trapped on follicular dendritic cells (FDCs) (Figure 2). FDCs are a non-hematopoietic stromal cell of specialized function that express complement receptors and Fcγ receptors and use these receptors to trap antigens and hold them on its cell surface. The light zone is where selection based on affinity occurs, and the selected cells migrate to the dark zone to somatically mutate their Ig genes, proliferate, and then migrate back to the light zone for another round of selection (Figure 2). While proliferating GC B cells are concentrated in the dark zone, accounting for its histological appearance, this distinction is not absolute, and so recently there has been a move away from the earlier nomenclature of referring to dark-zone GC B cells as “centroblasts” and light-zone GC B cells as “centrocytes”. Dark-zone and light-zone GC B cells can be distinguished reasonably well by flow cytometry by using relative changes in cell surface markers, including those that control the migration between the two zones, such as the chemokine receptor CXCR4, which attracts GC B cells to the dark zone and is more highly expressed on dark-zone GC B cells11.\n\nIn the light zone, GC B cells extract antigen (red polygon) from follicular dendritic cells (FDCs) in an affinity-dependent manner, internalize it into endosomes, partially degrade it into peptides (red triangles), and load those peptides onto major histocompatibility complex (MHC) class II molecules as in Figure 1. Follicular helper T (TFH) cells rapidly scan the B cells in the light zone and provide stimulatory signals (CD40L and cytokines as in Figure 1) to those B cells that present the most antigen. These signals promote the survival of B cells and also induce c-Myc, promoting proliferation in the dark zone, which is preceded by somatic hypermutation of immunoglobulin gene variable regions by activation-induced cytidine deaminase. After several rounds of proliferation in the dark zone, the mutated clonal progeny migrate back to the light zone and compete for uptake of antigen and receipt of T-cell help. These cycles of mutation, clonal expansion, and selection repeat for weeks to months, as long as the GC response continues. Memory B cells are generated throughout the GC reaction, whereas long-lived plasma cells are preferentially generated from high-affinity GC B cells late in the response. Abbreviations: BCR, B-cell antigen receptor; TCR, T-cell receptor.\n\nThe dark-zone and light-zone subcompartments of the GC appear to promote efficient affinity maturation but are not absolutely required. For example, deletion of the gene encoding CXCR4 in GC B cells prevents migration from the light zone to the dark zone22 and compromises the efficiency of selection for higher-affinity B cells, but some improvement in affinity still occurs. Interestingly, the distinctive properties of dark-zone GC B cells still are observed in a subset of CXCR4−/− GC B cells, indicating that cues from the microenvironment of the dark zone are not required to induce the dark-zone phenotype but rather the phenotypic changes are part of a cell-intrinsic program initiated in the light zone23. Interactions of higher-affinity GC B cells with TFH cells in the light zone are thought to promote their survival via the Bcl2-family member Mcl124, induce changes in expression of chemoattractant receptors to promote migration back to the dark zone (i.e., CXCR4), and induce expression of the key cell cycle regulator c-Myc25,26, leading to several rounds of proliferation27.\n\nEarly studies of the accumulation of mutations and increased affinity during the antibody response concluded that the GC response involves multiple rounds of mutation, cellular proliferation, and selection, resulting in increased affinity over time, a conclusion supported by recent intravital imaging studies11,28. As mentioned above, each round of mutation and selection is coupled with migration to the dark zone and back to the light zone. The mechanism of affinity selection in the GC is still an area of active investigation, but the following model is reasonably well supported by experimental data11. Following a short period of somatic mutation by AID and several rounds of cell division in the dark zone, the GC B cell enters the light zone and uses its BCR to extract antigen from the surface of FDCs. It is believed that the affinity of the GC B cell for antigen determines how much antigen it can extract from the FDC (see below). The antigen internalized by a GC B cell is processed into peptides, which are loaded onto MHC class II molecules and trafficked to the cell surface for recognition by TFH cells. The TFH cells actively migrate within the light zone of the GC, where they form relatively short-lived (minutes in duration) associations with antigen-presenting B cells19. Thus, TFH cells are sampling many different GC B cells, and evidently this broad sampling allows the TFH cells to calibrate their response such that they form the longest contacts with those GC B cells expressing the greatest number of cognate peptide/MHC II complexes and thus provide the strongest survival and proliferation signal to the higher-affinity B cells27,29. BCR signaling induced by antigen may also promote survival of GC B cells, but it is probably not the limiting factor that shapes selection in the GC11. Rather, current evidence indicates that TFH cells provide the key selection signals that maintain GC B-cell survival and allow them to re-enter the dark zone and participate in a new round of mutation and clonal expansion, followed by migration back to the light zone and further selection (Figure 2).\n\nThe selective signals provided by TFH cells to GC B cells are delivered by cytokines, with interleukin-21 (IL-21) being especially important30,31, and by the cell-bound tumor necrosis factor (TNF)-superfamily member CD40L11. Blocking CD40L in mice with anti-CD40L antibodies at any time during a GC response leads to rapid dissolution of the GC11. Similarly, genetic defects in the X-linked CD40L result in X-linked hyper-IgM syndrome32–34, in which individuals make IgM but no class-switched isotypes and fail to make GC responses. A very similar clinical syndrome results from genetic deficiency in the autosomally encoded AID14. In addition, GC B cells express inducible T-cell co-stimulator ligand (ICOS-L), the ligand for ICOS, the inducible co-stimulatory molecule. ICOS-L provides important co-stimulation to TFH cells to increase the synthesis of IL-21 and CD40L. In the absence of ICOS, individuals have greatly impaired antibody responses and are included within an immunodeficiency category of diverse genetic causes called combined variable immunodeficiency35, which is similar to hyper-IgM syndrome in that the GC response is largely defective. The interactions of GC B cells and TFH cells also require adhesion molecules of the signaling lymphocytic activation molecule (SLAM) family that signal through the adaptor SLAM-associated protein (SAP), as indicated by defects in the GC response in the genetic immunodeficiency disease X-linked lymphoproliferative syndrome (XLP), caused by loss of function mutations in SAP36. In XLP, the ability of cytotoxic T cells to control the proliferation of Epstein-Barr virus-infected B cells is compromised, leading to the observed lymphoproliferation, but GC responses are also thought to be poor in these individuals36.\n\nAlthough the competition in the GC is primarily between B cells in the same GC, this process is influenced by the soluble antibody that has been secreted up to that point in the response. The antigen on the surface of FDCs has bound to it secreted antibody37, so the GC B cells must compete with this bound antibody to be able to extract antigen, providing one driver to enhance affinity on an epitope-by-epitope basis and representing a competition between different GCs. The ability of GC B cells to extract antigen from FDC likely involves interactions of the BCR with the cytoskeleton inside the GC B cells, permitting mechanical strength to be generated as part of the process38.\n\n\nImmune tolerance to self and the germinal center response\n\nSomatic hypermutation of antibody genes runs the risk of generating variants with increased reactivity to self-antigens1. Indeed, analysis of anti-nuclear antibodies from patients with lupus indicates that their affinity was substantially enhanced by somatic mutations, probably in GC responses39,40. Similarly, GC responses have been shown to be important for autoantibody production in some, but perhaps not all, mouse models of lupus41–43. One documented mechanism that purges self-reactive GC B cells is that their expression of Fas makes them susceptible to killing by FasL expressed by helper T cells44,45, although exactly how that distinguishes self-reactive B cells from B cells responding to the foreign antigen remains unclear. The inhibitory FcγRIIB has also been implicated as important for a B-cell tolerance checkpoint in the re-stimulation of memory B cells46.\n\nAs the receipt of help from TFH cells is thought to be the limiting factor in affinity selection, one would expect that GC B cells that have acquired increased specificity for self-antigens would still need to be able to bind and internalize the initiating antigen in order to present peptides to TFH cells and be selected to survive and expand. Nonetheless, it may be that in some cases there is sufficient cross-reactivity between a foreign antigen and a self-antigen that GC B cells with increased affinity for a self-antigen would be selected and become plasma cells secreting the autoantigen. Perhaps the best-documented example of this “molecular mimicry” model for autoimmune disease in humans is in Guillain-Barré syndrome, where some strains of Campylobacter make a carbohydrate that is very similar to gangliosides on peripheral neurons, and some of the people who experience a severe infection with the corresponding strains make anti-ganglioside antibodies that likely cause the resulting peripheral neuropathy47. Less is known about the mechanisms by which autoimmune responses are triggered for most other antibody-mediated autoimmune diseases, although as mentioned above there is evidence for the importance of somatic mutations for development of anti-DNA antibodies in lupus. The possible role of Toll-like receptors (TLRs) in this process is discussed below.\n\n\nAdjuvants promote the quality of the germinal center response\n\nBoth the magnitude of the antibody response and the degree of affinity maturation are strongly influenced by the adjuvants used in a vaccination48. TLRs have emerged as an especially important innate immune pathway for promoting the antibody response. Whereas an early study had indicated that TLR recognition by B cells could promote the antibody response when using a pure TLR ligand as the adjuvant49, another study found that mice doubly deficient for the two main TLR signaling adaptor molecules, MyD88 and TRIF, responded normally to immunization with standard adjuvants50, likely reflecting alternative innate immune pathways also stimulated by such adjuvants. A variety of other studies have clearly demonstrated that TLR ligands make excellent adjuvants51; indeed, one such ligand is currently approved for use in human vaccines52. Indeed, live attenuated viral vaccines are among the best vaccines in human practice53, and virus-like particles, in which nucleic acid ligands for TLR7 or TLR9 are present inside the particle54,55 or nanoparticles with antigen and TLR ligands attached48, also induce outstanding antibody responses.\n\nWhereas these studies established that TLRs can serve as adjuvants for antibody responses, some initial studies suggested that they did so by promoting a strong extrafollicular antibody response56. Subsequent studies, however, have made it clear that TLR recognition can dramatically enhance the GC response, both in its magnitude and in the degree of affinity maturation48,55,57. Ligand recognition by the TLRs of both DCs and GC B cells promotes the GC response, but in different ways57. DC recognition of nucleic acid ligands for TLR7 or TLR9 promotes the magnitude of the GC response and increases the overall amount of IgG specific for the antigen but does not enhance affinity maturation. The effect of recognition by DC TLRs is likely a reflection of stronger generation of TFH cells during the early part of the response. In contrast, GC B-cell recognition of TLR7 or TLR9 ligands has a minimal effect on the total amount of specific IgG produced but substantially enhances the quality of the GC response48,57, including affinity maturation, the number of memory B cells produced, and the isotype of IgG produced, favoring a more inflammatory isotype of IgG57. The ability of TLR7 or TLR9 in the antigen-specific B cell to enhance the GC response is dependent on the nature of the antigen; monomeric protein antigens, which have a limited ability to induce BCR signaling, poorly engage TLR7 or TLR9 on B cells and have little impact, whereas oligomeric haptenated-protein antigens with TLR ligands attached exhibit a several-fold enhancement in the response, and for highly repetitive virus particles, there is a dramatic positive effect on the GC response, and production of IgG increases up to 30-fold55. In mice, the ability of TLR7 or TLR9 in the B cell to respond to virus genomic material has been found to be critical to the generation of neutralizing antibodies and the ability to control virus infection for acute infection with Friend erythroleukemia virus58, the chronic version of lymphocytic choriomeningitis virus (LCMV clone 13)59,60, and endogenous murine leukemia viruses61. Thus, it is likely that the enhancement of the GC response by TLR7 or TLR9 action in B cells is an evolutionarily selected mechanism to aid in generation of high-affinity antibodies to defend against viruses. The molecular mechanisms of this enhancement of the GC response are the subject of ongoing efforts.\n\n\nToll-like receptors and autoantibody responses\n\nAlthough TLR7 and TLR9 in B cells promote immune defense against viruses by permitting efficient generation of neutralizing antibodies, the same pathway has been implicated in the production of pathogenic antibodies in the systemic autoimmune disease, systemic lupus erythematosus62. The ability of the BCR and TLRs to synergize for B-cell activation was first recognized in studies examining B-cell proliferation in vitro62. Subsequent studies showed that, in the MRL/lpr mouse model of lupus, TLR7 and TLR9 contributed critically to production of anti-ribonucleoprotein IgG antibodies and to production of anti-double-stranded DNA antibodies, respectively63. Moreover, the TLR signaling component MyD88 was shown to be required in both DCs and in B cells in the Lyn−/− mouse model of lupus43. In this mouse model, the GC response has been implicated, since the autoantibodies are absent if SAP is deleted, which affects primarily the GC response and not the extrafollicular antibody response36. Similarly, in a separate mouse model of lupus-like autoimmunity in which TLR7 expression is increased via a transgene, autoantibody production was dependent on the GC response and on TLR7 action in B cells64. In human lupus, affinity maturation is typically observed for the antibodies that recognize double-stranded DNA65. Noteworthy in this regard, both human and mouse B cells express TLR7 and TLR9. Thus, it appears likely that TLR7 or TLR9 in autoreactive B cells contributes importantly to promoting affinity maturation of autoantibodies, likely enhancing their pathogenicity. Whether this pathway can be targeted therapeutically in patients with lupus remains to be seen, but several companies are currently testing TLR antagonists and chemical inhibitors of IL-1 receptor-associated kinases (IRAKs), which are required signaling molecules recruited by the adaptor MyD88. Also of note in this regard is that often lupus is treated with hydroxychloroquine, which decreases the acidity of late endosomes, although this has not been proven to be effective in a randomized clinical trial. As the processing of TLR9 and TLR7 into their active forms involves acid-requiring proteases in endosomes66, the efficacy of hydroxychloroquine in patients with systemic lupus erythematosus may result- from inhibition of TLR7 and TLR9 activity.\n\n\nSummary\n\nThe GC response is a critical element of the antibody response that is responsible for the production of high-affinity antibodies, long-lived plasma cells secreting these high-quality antibodies, and the generation of a large number of diverse memory B cells to help jumpstart antibody responses to subsequent infections by related pathogens. Individuals with selective defects in the GC response exhibit a range of susceptibilities to various infections. Studies of such individuals have greatly informed our understanding of the genes and molecular pathways that are important for the GC response. Recent studies have begun to determine how innate immune pathways may enhance the GC response, which may have important applications in the development of new vaccines against challenging targets. In addition, some autoimmune diseases likely involve GC reactions to produce the autoantibodies responsible for disease pathology, so increased understanding of the molecular mechanisms underlying GC responses may inform therapeutic efforts for those diseases.",
"appendix": "Competing interests\n\n\n\nThe author declares that he has no competing interests.\n\n\nGrant information\n\nThis review was supported in part by National Institutes of Health grant R21AI117378-01.\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\nGoodnow CC, Vinuesa CG, Randall KL, et al.: Control systems and decision making for antibody production. Nat Immunol. 2010; 11(8): 681–688. PubMed Abstract | Publisher Full Text\n\nTarlinton D, Radbruch A, Hiepe F, et al.: Plasma cell differentiation and survival. Curr Opin Immunol. 2008; 20(2): 162–169. PubMed Abstract | Publisher Full Text\n\nAda G, Isaacs D: Carbohydrate-protein conjugate vaccines. Clin Microbiol Infect. 2003; 9(2): 79–85. PubMed Abstract | Publisher Full Text\n\nVictora GD, Wilson PC: Germinal center selection and the antibody response to influenza. Cell. 2015; 163(3): 545–548. 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}
|
[
{
"id": "13993",
"date": "25 May 2016",
"name": "David Tarlinton",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13994",
"date": "25 May 2016",
"name": "Ulf Klein",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-999
|
https://f1000research.com/articles/5-998/v1
|
25 May 16
|
{
"type": "Review",
"title": "Focusing super resolution on the cytoskeleton",
"authors": [
"Eric A. Shelden",
"Zachary T. Colburn",
"Jonathan C.R. Jones",
"Eric A. Shelden",
"Zachary T. Colburn"
],
"abstract": "Super resolution imaging is becoming an increasingly important tool in the arsenal of methods available to cell biologists. In recognition of its potential, the Nobel Prize for chemistry was awarded to three investigators involved in the development of super resolution imaging methods in 2014. The availability of commercial instruments for super resolution imaging has further spurred the development of new methods and reagents designed to take advantage of super resolution techniques. Super resolution offers the advantages traditionally associated with light microscopy, including the use of gentle fixation and specimen preparation methods, the ability to visualize multiple elements within a single specimen, and the potential to visualize dynamic changes in living specimens over time. However, imaging of living cells over time is difficult and super resolution imaging is computationally demanding. In this review, we discuss the advantages/disadvantages of different super resolution systems for imaging fixed live specimens, with particular regard to cytoskeleton structures.",
"keywords": [
"microscopy",
"live cell imaging",
"cytoskeleton"
],
"content": "Introduction\n\nVisualizing proteins indirectly in cells and tissues at the light microscopic level using antibodies conjugated to fluorochromes revolutionized the field of cell biology 40 years ago. In the late 1980s and early 1990s, the use of commercial, user-friendly, confocal microscopes in combination with digital image acquisition systems dramatically improved the image quality of fluorescently labeled specimens and made image capture easier by avoiding the vagaries of the dark room. The technical benefits of confocal over “conventional” microscopy include the removal of the out-of-focus glare that interferes with what the imager really wishes to observe and an increase in specimen contrast. More recently, the ability to follow tagged molecules in live cells using both conventional and confocal fluorescence microscopy and the development of molecular biosensors has allowed cell biologists to study the dynamics of individual proteins and protein complexes with high precision. Conventional techniques used for light microscopy can achieve resolutions of up to ~100 nm in the image plane, as first detailed by Abbe1, and about twice this value along the focal axis2. In practice, most authors consider the resolution of commercially available microscopes to be ~200 nm in the image plane and ~500 nm in the axial dimension. So called “super resolution” is achieved with techniques that allow resolution beyond the diffraction limit of conventional optics3. The term super resolution as applied to microscopy has been in use since at least as early as the 1960s4. Resonance energy transfer5, near field scanning optical microscopy6, dual objective (4Pi) microscopy7, total internal reflection microscopy8, single molecule fluorescence localization9, expansion microscopy10, and several other strategies represent successful efforts to obtain structural or positional information from biological specimens at resolutions better than those afforded by conventional microscopy. However, many of these approaches are technically demanding or present significant limits to the type of specimens that can be examined. The last decade has seen the development of methods that closely resemble more familiar far-field and laser scanning confocal microscopy but allow direct visualization of subdiffraction size structures in fixed and living specimens. Current super resolution techniques provide resolutions of less than 10 nm in the image plane11 and ~20 nm in the z-axis. The improved resolution offered by these methods has produced breathtaking images of the nuclear pore complex12,13, the tubular walls of microtubules14, and many other structures.\n\nAlthough the potential and allure of super resolution methods are indisputable, they also present new challenges to image acquisition, storage, and interpretation. For example, changing the resolution of an image from 200 nanometers to 20 nanometers in the image (xy) plane, while maintaining a fixed field of view, results in a 100-fold increase in image size. Extending these calculations to the third dimension, multispectral imaging and time (for live cells) reveals the potential extent of demands that super resolution microscopy can place on specimens and fluorophores as well as imaging and data processing hardware. Moreover, although computer and imaging technologies have advanced to the point where accumulating these data is feasible, human involvement is currently still needed to identify regions of interest for examination and analysis. Each of the methods used to achieve super resolution imaging also offer unique technical strengths and weaknesses to cell biologists. We direct the reader to several excellent recent reviews that provide an overview of the capabilities, advantages, and disadvantages of a variety of super resolution procedures in tabular form15–18.\n\nDespite the inherent challenges, the use of super resolution microscopy is beginning to make an impact on a wide variety of biological topics. Among the subjects most likely to benefit from the application of super resolution imaging is the study of the cytoskeleton and its associated structures. Unlike many other cellular components, cytoskeletal filaments form anastomosing networks of fibers smaller than the resolution of conventional imaging methods. Techniques allowing enhanced resolution imaging of cytoskeletal structures, especially in live cells, are already advancing our knowledge of their formation and function. For example, super resolution has been a boon to investigators studying cytoskeletal rearrangements in bacteria19,20 and yeast cells21, which have generally been too small to approach with conventional diffraction-limited imaging methods. Below, we briefly discuss the major methods for achieving super resolution as well as their strengths and weaknesses with emphasis on recent work in which these methods have been applied to address biological questions involving the cytoskeleton.\n\n\nLocalization microscopy\n\nStochastic optical reconstruction microscopy (STORM22), photoactivated localization microscopy (PALM23), fluorescence photoactivation localization microscopy (FPALM24), and a growing number of related methods are techniques where fluorescent specimens are examined by activating a limited set of fluorophores at a time which must be separated by distances greater than the resolution limit of the microscope. A diffraction-limited image of the fluorophores is captured and the position and intensity of each fluorophore calculated at subdiffraction precision. Activated fluorophores are deactivated, and a new set of fluorophores is activated and imaged. After many images are collected, a completed super resolution image is calculated. The techniques differ in the type of fluorophore used. For example, PALM and FPALM are used to image expressed photoactivatable fluorescent proteins and fusion proteins23,24, while STORM is used to create images of fluorescent dyes and tags that can be switched between fluorescing and non-fluorescing states22. In practice, these methods can produce the highest resolution of the available diffraction-unlimited techniques when applied to biological specimens, with two-dimensional resolutions of less than 20 nm frequently reported12,25–27. However, overall image resolution and quality increase with the number of photons captured and fluorescent molecules examined. Therefore, these methods are limited by the time required to obtain a sufficient number of images – often tens of thousands – needed to create a final super resolution image. In addition, many conventional fluorophores and fluorescent proteins are not suitable for localization microscopy14,28, making some techniques such as multicolor staining methods challenging. Localization methods also require that fluorescent molecules or proteins within the specimen be detected individually, complicating the application of these techniques to densely labeled three-dimensional cytoskeletal arrays or arrays contained within thick samples exhibiting autofluorescence. To address this, some investigators have incorporated total internal reflection fluorescence illumination (TIRF), two photon illumination, or light sheet illumination to limit the volume of a specimen under inspection23,29–31. However, these implementations increase the complexity of the instrumentation required. Despite these limitations, a large number of studies encountered in our review of current literature employ localization techniques. This may be because of both the superior resolution of the methods and the relatively simple hardware requirements which have allowed many investigators to build their own STORM or PALM/FPALM imaging systems. In addition, fluorophores, fluorescent proteins, buffering agents, and illumination strategies are being actively developed to extend localization to both multicolor labeling and other fluorescent imaging tasks (see 28,32–35 for representative reviews).\n\nAmong the most novel and dramatic discoveries made using localization methods is the periodic distribution of actin filaments and associated cytoskeletal proteins in axons first visualized using STORM imaging36. The periodicity of this structural feature of neurons is below the resolution limit of conventional microscopy and was overlooked in thin section electron micrographs37,38. Other studies have further exploited single molecule/protein imaging to analyze the development and regulation of these arrays39,40. STORM and PALM methods are also being applied to the study of the structure of adhesion complexes such as hemidesmosomes41, intercellular adherens junctions42,43, and the less well-defined adhesions formed by leucocytes44, as well as actin cytoskeletal rearrangements that occur during endocytosis45 and bacterial host cell invasion46. In the case of adherens junctions, STORM analyses indicate that E-cadherin exists in clusters at sites of cell-cell interaction rather than as the “solid” belt typically observed by non-super resolution methods42,43. Similarly, dual-color PALM studies suggest that paxillin and vinculin form functionally distinct non-overlapping nanoaggregates in focal adhesions that are not detectable using conventional imaging methods47.\n\nMicrotubules, 24 nm diameter tubes composed of protofilaments, are sparsely distributed at the edge of cultured cells and are often used as proof-of-concept targets by developers of super resolution imaging methods48–51. STORM and PALM imaging are beginning to provide new details regarding the function and organization of the microtubule cytoskeleton. For example, these techniques have been used to study the organization of centrosomal proteins in intact cells, the architecture of microtubules underlying the movement of organelles within living cells52, and the interaction of kinesin motor proteins with microtubules in neuronal processes53. A variant of PALM imaging has also been used to study the structure of EB1 at the distal tip of growing microtubules54, and PALM has made possible the visualization of FtsZ, the bacterial homolog of eukaryotic tubulin, in distinct polymeric arrays in prokaryotes20,55.\n\nAlthough relatively few studies have examined intermediate filament arrays using super resolution imaging, STORM has been used to investigate keratin, plectin, and integrins in hemidesmosomes formed by cultured keratinocytes41. Additionally, desmin, a cytoskeletal protein mutated in clinically important cardiomyopathies, has been visualized using dual color PALM microscopy in cultured cardiomyocytes56. In the latter study, the authors report a 10-fold increase in the resolution of desmin protein aggregates and filaments over non-super resolution light microscopic methods. More importantly, their super resolution images reveal that both mutant and wild-type desmin proteins are incorporated into the same filament, suggesting the possibility that changes in the mechanical properties of a “mixed” filament might be the cause of disease56.\n\n\nStructured illumination microscopy\n\nStructured illumination microscopy (SIM) improves the resolution of light microscopy by illuminating a specimen with a defined regular pattern of diffraction-limited light and dark bands which create Moiré patterns when combined with the structure of a specimen57. An image of the resulting interference pattern is created and recorded. The illuminating pattern is then rotated and further images captured. Finally, an image with improved resolution is calculated from the combined rotation series. Because the illuminating pattern is generated using wide-field optics, the technique exposes a specimen to illumination levels that are comparable to other wide-field microscopy methods (although multiple images must be captured for each view of the specimen). While many implementations of localization microscopy use total internal reflection illumination and are therefore limited to observation of structures within ~100 nm of an optical surface, SIM can resolve structures many microns deep within a specimen. SIM can also be used with any fluorescent probe and, unlike localization methods or laser scanning methods such as stimulated emission depletion microscopy (STED), complete images of a specimen are obtained at speeds determined by camera sensitivities. SIM is therefore one of the least phototoxic and most rapid methods for obtaining enhanced resolution images and has been used extensively in studies of living cells. A three-dimensional version of structured illumination allows for improved resolution in the z direction and has been used to visualize cytoskeletal structures in three dimensions. Both of these applications are discussed below.\n\nThe resolution achieved with SIM is generally only twofold greater than that offered by conventional microscopy57, although some implementations allow SIM to achieve lateral resolutions as high as 50 nm58,59. However, even a twofold increase in resolution, combined with the power of multispectral fluorescent labeling methods, has allowed investigators to observe previously unresolved features of a wide variety of cytoskeletal structures. For example, SIM has been used to characterize the distribution of microtubules and associated structures in a variety of specimens that conventional diffraction-limited imaging methods have been unable to resolve well, including the neuromuscular junction60, platelets61, centrosomes62,63, and protists64. SIM has also facilitated studies investigating the structure of striated muscle65,66 as well as actin and myosin filament organization in non-muscle cells67–70. Intermediate filament architecture and associated junctions have also been examined using this method71,72. Interestingly, although the localization-based imaging methods described above achieve higher resolution than SIM, the pointillized appearance of images produced by localization microscopy can obscure fine structural detail that may be visible using SIM. For example, SIM has revealed that focal adhesions comprise linear subarrays73, a feature not readily visible in images published by investigators using interferometric (i)PALM to analyze the axial distribution of focal adhesion components74–76.\n\nFinally, because SIM can be applied to microscopy of any fluorescent probe, it can be readily used for multispectral studies using conventional fluorophores. SIM has been used to examine cytoskeletal structures in triple-labeling studies of adherens junctions77,78, neuronal spines79, centrioles80, kinetochores81, and endosomal vesicles82. For example, SIM analyses of VASP, zyxin, testin, and other proteins surprisingly indicate that these tension-regulating proteins are likely recruited to adhesion junctions independent of the core adhesion complex78.\n\n\nStimulated emission depletion microscopy\n\nSTED, reversible saturable optical fluorescence transitions microscopy (RESOLFT), and related techniques excite fluorophores in a diffraction-limited spot by a focused laser. Outlying fluorophores are converted to a non-fluorescent state by illumination with a second (depletion) laser in a manner that leaves a central, subdiffraction limited area of fluorophores unconverted83–85. The remaining still-fluorescent fluorophores are detected to create an image with resolutions far greater than those provided by conventional imaging methods. Some implementations of these techniques have yielded resolutions in biological specimens of <50 nm in the image plane and 150 nm in the axial dimension86. Common implementations of STED are technically demanding and the depletion light energies are substantially greater than the illumination intensities required by other super resolution imaging methods. However, STED and similar systems closely resemble laser scanning confocal microscopes already employed by many investigators, and these methods can be applied to most commonly available fluorophores. Unlike localization methods and SIM, STED does not require calculations to generate enhanced resolution images, and image resolution can be readily varied by changing the raster scanning patterns used to visualize a specimen. Scanning rates achieved by STED are suitable for imaging of live specimens87,88, but the required depletion energies currently make extended live cell imaging a challenge. However, super resolution imaging can also be interchanged with conventional confocal scanning approaches simply by turning the depletion laser on or off.\n\nSTED has been used to visualize the periodic ring structure of actin in neurons89 and actin dynamics in dendritic spines of neurons in living brain tissue90. Others have used STED to examine the actin-like MreB protein in bacteria91 and the reorganization of actin filament arrays during activation of natural killer cells and T cells92,93. It has allowed the visualization of myosin mini-filament formation in mammalian non-muscle cells94 and insight into the regulation of the actin cytoskeleton by intracellular signaling proteins95,96. Microtubules have been examined using STED in primary cilia97, and the interphase microtubule array has been examined in muscle and non-muscle cells98,99. Finally, several groups have utilized STED microscopy to visualize elements of the intermediate filament network, including vimentin100,101, keratin102, and nuclear lamins103. In our laboratory, we have used STED to assay the relative localization of vimentin and focal adhesions in cultured epithelial cells. Whether vimentin interacts with focal adhesions has been controversial for years104,105. However, STED not only provides images with more detail than can be obtained using conventional confocal microscopy but also reveals a distinct pattern of organization of paxillin within a focal adhesion (Figure 1). Moreover, in the STED image, vimentin filaments clearly wrap around each focal adhesion, an interaction that is not apparent in the confocal image.\n\nConventional confocal imaging is shown in A while STED imaging of the same area is shown in B.\n\n\nLive cells\n\nThe ability to study proteins in cells in the living state is one of the most significant advantages of light microscopy over other methods of analysis. However, as has been noted by many others (see 106–108 for representative reviews), obtaining images of living cells that are doing something other than dying on the microscope is difficult and involves balancing the competing requirement of image quality and cell health. In addition, the study of cytoskeletal dynamics often requires rapid image acquisition, increasing the light exposure of live cells over time. Achieving resolutions greater than the diffraction limit of conventional microscopy ultimately requires capturing larger numbers of photons from smaller areas of live cells than is necessary for conventional resolution images, an effort that can clearly compromise the integrity of cells under examination108. Fluorescently labeled cytoskeletal filaments are also notoriously photolabile109. To date, many investigators of the cytoskeleton in live cells have combined super resolution microscopy of fixed cells with wide-field or confocal imaging of live cells39,44,45,52,53,64,68,69,77,79,98,110–113 or have obtained single images of live cells using super resolution methods56,89. Although the latter avoid potential artifacts induced by fixation and immunostaining, this approach does not address dynamic changes in cell architecture.\n\nThe majority of publications in our survey that examined dynamic changes in cytoskeletal architecture employed SIM. Observations of the cytoskeleton in live cells have been made over time scales ranging from seconds to tens of minutes using this approach. The twofold resolution enhancement offered by SIM has allowed investigators to examine in previously impossible detail the growth of microtubules in living plant cells114, actin retrograde flow in cultured insect cells115, and the reorganization of cytoskeletal arrays in dividing yeast and bacterial cells21,91. Two-color applications of SIM in living cells have been used to demonstrate heterotypic assembly of myosin II isoforms67, the spatial relationship between myosin IIA and alpha actinin116, the clustering of receptors in adherens junctions117, and that vimentin intermediate filaments move bi-directionally along microtubules118.\n\nAlthough capturing clear images of closely apposed structures using localization methods requires accumulation and processing of thousands of images over periods of many seconds to minutes, localization methods have also been applied to the study of rapidly changing cytoskeletal structures in live cells by sacrificing some image clarity to improve temporal resolution. For example, PALM has been used to quantify the addition and loss of individual paxillin proteins at focal adhesion sites with a spatial resolution of 60 nm and temporal resolution of 25 seconds119. Proof-of-principle studies have also demonstrated that PALM can be used to visualize dynamic changes in fluorescent actin filaments over brief intervals120 and the movement of fluorescently labeled endosomes along microtubules in living axons121. In addition, PALM imaging platforms have been exploited by several groups that have followed changes in localization of single fluorescent cytoskeletal proteins over time, either alone or in combination with PALM, STORM, or STED techniques122–125. This appears to be a powerful, multimodal approach that can place the movement of individual proteins in the context of cytoskeletal architecture.\n\nSTED employs depletion lasers at energies that are largely incompatible with extended viewing of live cells, and, not surprisingly, we encountered few publications where this method has been used to image cytoskeletal arrays in vivo. Nonetheless, STED has great potential for imaging structures within complex tissues and has been used in a seminal study of actin dynamics in living neurons within brain tissue90. Single images of microtubules within living cells have also been obtained at 60 nm resolution using STED86. STED has also been applied to imaging using multiphoton excitation126–128 and total internal reflection microscopy129. These methods limit light exposure to a sample and offer further potential for the application of STED illumination to live cells. We anticipate that as these complex instruments become more widely available, we will see an increase in the number of investigators employing them for studies of living cells.\n\n\nThe third dimension\n\nMany initial implementations of super resolution methods did not provide an increase in resolution in the third, or axial, dimension of the microscope (see 18 for review). However, recent modifications to these methods have achieved impressive resolution enhancements in the third dimension. Three-dimensional SIM doubles the resolving ability of the light microscope in all dimensions while retaining its ability to obtain images rapidly with low light exposures130. Although many studies have captured Z-stacks of images using super resolution methods and generated extended focus images from them, relatively few studies have analyzed the cytoskeleton in three dimensions using super resolution microscopy. However, three-dimensional SIM has been used to resolve actin filament arrays and microtubules in three dimensions using both fixed and live cultured cells113,116,130,131 as well as to study the structure of centrosomes63,80 and kinetochores81. Additionally, this method has also been used to visualize the three-dimensional organization of FtsZ in dividing bacteria132. Modification of STORM and PALM imaging platforms can achieve axial resolution of up to 20 nm by introducing axial astigmatism into the optical path133 and by generating interference patterns from images obtained with paired, opposed objective lenses134. Three-dimensional STORM and PALM have been used to study the movement of organelles along microtubules in live cells and the formation of FtsZ ring structures in live dividing bacteria20,52. A STORM imaging method employing axial astigmatism and dual objectives has been used to show that sheet-like cellular extensions in cultured cells contain two distinctly separate actin filament arrays each with unique patterns of actin filament organization134. The development and regulation of these previously undetected cytoskeletal arrays have been further analyzed using similar approaches in normal cell movement112,116. iPALM can resolve structures less than 20 nm in diameter in three dimensions27. The technique has recently been used to dissect the organization of cytoskeletal proteins associated with matrix adhesion devices termed focal adhesions at an unprecedented level of detail74–76. Impressively, the technique was able to resolve not only vertical stratification enriched for specific cytoskeletal components but also the polarized orientation of the N- and C-terminal ends of talin within focal adhesions of intact cells. Finally, STED microscopy has also been modified by the addition of dual depletion patterns, one oriented in the image plane and the other in the axial dimension, allowing for increases in resolution in the third dimension of up to 125 nm135. STED and RESOLFT have also been extended to the third dimension using a dual objective imaging strategy136.\n\nPresently, most work using super resolution imaging has been conducted using single cells cultured on optical surfaces. However, previous work has shown that cell morphology and behavior can be dramatically altered in three-dimensional environments137,138. To date, the literature examining the cytoskeleton in cells in situ is largely limited to proof-of-principle studies. However, these efforts demonstrate the growing potential of super resolution methods. For example, STED has been used to view the dynamics of actin filament arrays within live neurons in 350 μM thick brain slices90, and three-dimensional SIM has been used to visualize actin arrays in developing Drosophila115. In other studies, labeled nuclear histones have been visualized in cells within 150 μM cell spheroids by combining single molecule localization methods with planar illumination139. Both planar and multi-photon illumination methods have been coupled with structured illumination to view green fluorescent protein (GFP) expressed in living nematodes140,141. These methods improve visualization by restricting effective illumination to a single plane of interest within thick specimens. While these latter studies examined structures other than the cytoskeleton, these methods show promise for the analysis of the cytoskeleton at subdiffraction resolutions in situ.\n\n\nSummary\n\nMany of us have been fortunate to work as cell biologists during two major revolutions in imaging technology: the development of fluorescent proteins as tools for biologists and the development of confocal microscopy, which extracts clear, in-focus images in which the contaminating blur of out-of-focus structures has been removed. The astonishment and wonder with which we now view images created by super resolution microscopy suggest that we are experiencing yet a third revolution in the technology available to cell biologists. It is likely that for dual and triple labeling studies of cultured cells in two and three dimensions, as well as studies of bright, relatively slow-moving structures in live cells, super resolution imaging will soon replace confocal microscopy as the state of the art in much the same way that confocal microscopy replaced conventional wide-field imaging in the 1980s and 1990s. However, each of the approaches used to achieve images at better than diffraction-limited resolution have strengths and weaknesses. Studies of rapid cytoskeletal dynamics in live cells and three-dimensional studies are likely to present technical and biological challenges to practitioners of super resolution microscopy for some time. In addition, while the best super resolution light microscopic methods achieve resolutions of <10 nm, this is still 50–100-fold greater than the resolution afforded by electron microscopy142,143. Conventional fluorescence microscopes can also take advantage of a myriad of probes for physiological conditions and molecular interactions that have yet to be adapted to super resolution imaging methods. For the time being, no method addresses all possible experimental needs, and investigators will likely have to address the limitations of their super resolution instruments with complementary approaches involving conventional methods. Moreover, the dream of seeing individual protein complexes and their partners functioning in live cells within a complex three-dimensional organism remains unrealized. Nonetheless, we are seeing the development of instrumentation, computational methods, fluorescent probes, and novel methods at an amazing pace. We can only imagine what the next advances will be.",
"appendix": "Author contributions\n\n\n\nEric A. Shelden wrote the majority of the text with the assistance of Jonathan C.R. Jones. Jonathan C.R. Jones and Zachery T. Colburn edited the text. Zachery T. Colburn provided the images and processed cells for localization.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work was supported by grants from the National Science Foundation (IOS 1457368) to EAS and National Institutes of Health (AR054184) to Jonathan C.R. Jones. Zachery T. Colburn was supported, in part, by a Poncin Scholarship.\n\n\nReferences\n\nAbbe E: Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Archiv f mikrosk Anatomie. 1873; 9(1): 413–8. Publisher Full Text\n\nFrancon M: Phase-contrast microscopy. Progress in Microscopy. New York: Row, Peterson & Co, 1961.\n\nKolobov MI: Quantum Imaging. New York, NY: Springer New York; 2007. 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}
|
[
{
"id": "13981",
"date": "25 May 2016",
"name": "Patricia Wadsworth",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13980",
"date": "25 May 2016",
"name": "Jeffrey Segall",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13982",
"date": "25 May 2016",
"name": "Omar Skalli",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-998
|
https://f1000research.com/articles/5-996/v1
|
25 May 16
|
{
"type": "Research Note",
"title": "Spanish translation and preliminary validation of the Pain Treatment Satisfaction Scale",
"authors": [
"Teresa A. Nava-Obregon",
"Daniel A. Fragoso-Estrada",
"Sandra Castillo-Guzman",
"Norma G. Lopez-Cabrera",
"Omar González-Santiago",
"Dionicio Palacios-Rioas",
"Teresa A. Nava-Obregon",
"Daniel A. Fragoso-Estrada",
"Norma G. Lopez-Cabrera",
"Omar González-Santiago",
"Dionicio Palacios-Rioas"
],
"abstract": "Satisfactory pain treatment could prevent adverse physical and psychological outcomes in patients. In this sense, adequate survey to measure patient satisfaction of pain management is important. In this study, we translated the Pain Treatment Satisfaction Scale to Spanish and analyzed the psychometric properties of its five dimensions. Reliability was determined with Chronbach´s-α and convergent and divergent validity with a multitrait-multimethod analysis. The latter were reported as successful percentage. In the five dimensions, the reliability of the Spanish version showed Chronbach´s-α values ≥ 0.75; successful percentage for convergent validity was ≥ 88, and successful percentage for divergent validity was ≥ 94. The Spanish version showed good psychometric properties and can be used to measure pain treatment satisfaction.",
"keywords": [
"Treatment satisfaction",
"Pain treatment",
"PSTT",
"Mexico"
],
"content": "Introduction\n\nSatisfactory pain management is important to prevent adverse physical and psychological results for patients and their families. Through the activation of the pituitary-adrenal axis, inadequate pain treatment could produce postsurgical infection and poor wound healing. Pain also actives the sympathetic nervous system, which results in disruption of the cardiovascular, gastrointestinal and renal systems1. In addition, patients with pain may have a sense of helplessness or hopelessness. This may predispose them to anxiety and depression, and they may be reluctant to seek medical care for other health problems2.\n\nAdequate pain control can improve the patient’s attitudes and behaviors toward their treatment. They are more willing to comply with the advice of their health care provider, miss fewer medical appointments and are more likely to adhere to their medical treatment3. In this sense, surveys to measure patient satisfaction and pain treatment are important for adequate health care. The aim of this study was to translate the Pain Treatment Satisfaction Scale (PTSS)4 into Spanish and analyze some of its psychometric properties.\n\n\nMethods\n\nSurvey. The PTSS was developed by Evans et al.4 and validated to assess satisfaction in patients with both acute and chronic pain. This survey includes 39 items grouped into five dimensions: information about your pain and treatment (5 items); medical care (8 items); impact of current pain medication (8 items); satisfaction with pain medication, which included the two subscales medication characteristics (3 items) and efficacy (3 items); and side effects (12 items). Each item is measured with a 5 point-Likert scale which ranges from 1 (major satisfaction) to 5 (worst satisfaction). Scales and items were transformed to a 0 to 100 score, where 100 represents highest satisfaction.\n\nTranslation. This process was carried out in 4 steps. In the first step, 2 authors, experts in pain medicine (TANO and SCG), performed the translation from English to Spanish. In the second step, all authors reviewed the translation and gave suggestions and recommendations to improve the clarity of the items. In the third step, external professional translators made the inverse translation from Spanish to English. In the final step, all authors reviewed both the inverse translation and the original survey.\n\nSample. We applied the Spanish version to a random sample of 174 patients from the Dr José E. Gonzalez University Hospital with some type of pain. They were interviewed in the postoperative or ambulatory areas. Patients older than 18 years who gave verbal consent were included in this study. The Ethical Committee of the Faculty of Medicine of the Autonomous University of Nuevo León approved this study, and exempted from written informed consent. The reference number is AN15-004.\n\nStatistical analysis. Results of each dimension were calculate. Reliability was measured with Chronbach´s alpha test. The criterion of acceptability was 0.7. The construct validity, which included convergent and divergent validity, was evaluated with a multitrait-multimethod analysis. Convergent validity and divergent validity were expressed as %successful. Convergent validity was calculated with the following formula %successful = [(correlations “item–dimension to which it belongs >0.4”)/(total correlations “item–dimension to which it belongs”)]*100. The formula for divergent validity was %successful = [(number of correlations item – dimension lesser that correlations item-dimension to which it belongs)/(total correlations item-dimension to which it does not belong)]*100. Correlations were obtained with the matrix multitrait-multimethod. The statistical software SPSS 21 and the package Psy for R 3.2.2 were used for the analyses.\n\n\nResults\n\nThe final Spanish version of the PTSS is available as supplementary material. Of the patients surveyed, 53.5% were men and 46.5% were women. Overall, mean age was 41.5 years, and according to gender, the mean age of women was 38.5 years and for men 44.6 years. The type of pain identified was acute in 60.8% (postoperative 15.2%) and chronic in 39.2% of patients.\n\nResults of the PTSS are presented in Table 1. In general, patients with mild pain in the last 24 hours had a better score than patients with moderate or intense pain in almost all dimensions, except “side effects”.\n\nVAS scale for pain mild = 1 – 3; moderate = 4 – 7; intense = 8 – 10\n\n*From Evans 20043\n\n* From Evans 20043\n\nReliability and construct validity. The range of Chronbach´s alpha was 0.75 to 0.90, with medical care being the dimension with the lowest score and “impact of current pain medication” the dimension with the highest score. The %successful in convergent and divergent validity ranged from 88 to 100% and from 94 to 100%, respectively. “Medical care” (88%) and “side effects” (92%) were the dimensions with the lowest %successful in convergent validity, while medical care (94%) was the dimension with the lowest %successful in discriminant validity.\n\n\nDiscussion\n\nIn this study, we translated and preliminarily evaluated the psychometric properties of PTSS in its Spanish version. In other studies, this survey has been translated and validated in Chinese3 and French4 where good psychometric properties have been shown.\n\nIn the case of reliability, values obtained are satisfactory since values of all dimensions were > 0.70 and similar to the original version. In general, the values of all dimensions were slightly lower than the original version.\n\nWith respect to convergent and discriminant validity, results are satisfactory and similar to the original version. The medical care dimension had lower values than the original in both convergent and discriminant validity. Side effects had a value slightly higher than the original. The rest of the dimensions had the same values as those of the original version.\n\nThe limitations of this study include lack of a rigorous diagnosis of the type of pain. The sample size was smaller than that used in other studies3–5 and finally, a test-retest analysis was not performed.\n\n\nConclusion\n\nThe Spanish version of PTSS has satisfactory psychometric properties and some of these are similar to the original version; therefore it could be considered a valid instrument to measure pain treatment satisfaction in a Mexican population.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for ‘Spanish translation and preliminary validation of the Pain Treatment Satisfaction Scale’, 10.5256/f1000research.8750.d1227916",
"appendix": "Author contributions\n\n\n\nTANO and SCG conceived the study, DAFE an NGLC carried out the research, OGS and DPR prepared the first draft of the manuscript and performed the statistical analysis. 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\nAcknowledgments\n\nSergio Lozano Scientific Publications Support Coordinator Office of the Vice Dean of Research\n\n“Dr. Jose Eleuterio Gonzalez” University Hospital, Universidad Autónoma de Nuevo León, Monterrey, N.L., Mexico.\n\n\nSupplementary material\n\nSpanish version of the PTSS. Click here to download the file.\n\n\nReferences\n\nWells N, Pasero C, McCaffery M: Improving the Quality of Care Through Pain Assessment and Management. In: Hughes RG, editor. Patient Safety and Quality: An Evidence-Based Handbook for Nurses. Rockville (MD): Agency for Healthcare Research and Quality (US); 2008. PubMed Abstract\n\nHirsh AT, Atchison JW, Berger JJ, et al.: Patient satisfaction with treatment for chronic pain: predictors and relationship to compliance. Clin J Pain. 2005; 21(4): 302–310. PubMed Abstract\n\nWong WS, Chen PP, Chow YF, et al.: The Reliability and Validity of the Cantonese Version of the Pain Treatment Satisfaction Scale (ChPTSS) in a Sample of Chinese Patients with Chronic Pain. Pain Med. 2015; 16(12): 2316–2323. PubMed Abstract | Publisher Full Text\n\nJouini G, Choinière M, Dion D, et al.: Programme ACCORD: validation of a French version of the pain treatment satisfaction scale in primary care patients with non-cancer chronic pain [abstract]. Annual conference of the Canadian Association for Population Therapeutics; March 28–30, 2010; Toronto, Canada. Can J Clin Pharmacol. 2010; 17(1): e117, 58. Reference Source\n\nEvans CJ, Trudeau E, Mertzanis P, et al.: Development and validation of the Pain Treatment Satisfaction Scale (PTSS): a patient satisfaction questionnaire for use in patients with chronic or acute pain. Pain. 2004; 112(3): 254–266. PubMed Abstract | Publisher Full Text\n\nNava-Obregon TA, Fragoso-Estrada DA, Castillo-Guzman S, et al.: Dataset 1 in: Spanish translation and preliminary validation of the Pain Treatment Satisfaction Scale. F1000Research. 2016. Data Source"
}
|
[
{
"id": "13978",
"date": "11 Jul 2016",
"name": "Frédérique Servin",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper \"Spanish translation and preliminary validation of the Pain Treatment Satisfaction Scale\" evaluates the validity of an already published pain treatment satisfaction scale, presented in a Spanish translation, on a Mexican population. After a careful double checked translation, the paper concludes that it is adequate for use in the pre-defined population.\nThis paper is published as a Research note, and indeed, considering the number of items included in the score (39), it can hardly be proposed in clinical practice for repeated measures.\nIf the quality of the translation has obviously been well controlled, it is not the case for the study population. No estimation of the number of subjects required to demonstrate the validity of the score has been made, despite the fact that the authors had comparators against which they could perform this estimation. 174 appears as a very low number for a population including both acute and chronic pain in a vast variety of patients. Indeed, if the translation is accurate, any difference in its validity will come mainly from socio-cultural factors in the population. The authors themselves are indeed conscious of this limitation, but they could have easily corrected it.",
"responses": []
},
{
"id": "16266",
"date": "14 Sep 2016",
"name": "Richard Fielding",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI’ve looked at the paper and generally it reads well, and doesn’t seem to have many major problems methodologically, other than the fact that normally, for convergent and divergent validity, different scales that measure similar and dissimilar dimensions are used.\nWhat the authors refer to as divergent and convergent validity in their manuscript when they compare the Spanish and English versions. This is simply the concordance between the scores of English and Spanish instruments, which gives between 88%-100% concordance. This simply might reflect language proficiency effects. I would suggest, therefore that the authors choose some other term for this than convergent and divergent validity as this is incorrect.",
"responses": []
}
] | 1
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https://f1000research.com/articles/5-996
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https://f1000research.com/articles/5-992/v1
|
24 May 16
|
{
"type": "Review",
"title": "Dynamical systems, attractors, and neural circuits",
"authors": [
"Paul Miller"
],
"abstract": "Biology is the study of dynamical systems. Yet most of us working in biology have limited pedagogical training in the theory of dynamical systems, an unfortunate historical fact that can be remedied for future generations of life scientists. In my particular field of systems neuroscience, neural circuits are rife with nonlinearities at all levels of description, rendering simple methodologies and our own intuition unreliable. Therefore, our ideas are likely to be wrong unless informed by good models. These models should be based on the mathematical theories of dynamical systems since functioning neurons are dynamic—they change their membrane potential and firing rates with time. Thus, selecting the appropriate type of dynamical system upon which to base a model is an important first step in the modeling process. This step all too easily goes awry, in part because there are many frameworks to choose from, in part because the sparsely sampled data can be consistent with a variety of dynamical processes, and in part because each modeler has a preferred modeling approach that is difficult to move away from. This brief review summarizes some of the main dynamical paradigms that can arise in neural circuits, with comments on what they can achieve computationally and what signatures might reveal their presence within empirical data. I provide examples of different dynamical systems using simple circuits of two or three cells, emphasizing that any one connectivity pattern is compatible with multiple, diverse functions.",
"keywords": [
"hidden Markov model",
"Point attractors",
"Marginal states",
"line attractors",
"continuous attractors",
"Oscillating systems",
"cyclic attractors",
"limit cycles",
"strange attractors",
"Heteroclinics"
],
"content": "Introduction\n\nWhen we try to understand any biological process, our models of the system matter. Our ideas of how a parameter impacts the system or how a variable responds to manipulations of the system determine what questions we attempt to answer and which experiments we perform. These ideas are based on our own mental models, which can be misleading if not founded on the appropriate dynamical principles1,2.\n\nIn this article, I hope to provide a short introduction to the types of dynamical system that arise in neural circuits. In doing so, I explore why there is a perhaps surprising lack of consensus on the nature of the dynamics within mammalian neural circuits—modelers aiming to explain various cognitive processes commence from seemingly incompatible starting points, such as chaotic systems, oscillators, or sets of point attractor states. I briefly consider hallmarks and support for each paradigm and summarize how—in spite of the tremendous quantity of electrophysiological data—room for debate remains as to which type of dynamical system is best used to model and understand brain function.\n\n\nHidden variables within neural data\n\nNeural circuits are nonlinear dynamical systems that, in principle, can be described by coupled differential equations3. However, the relevant continuous variables necessary for a full description of the behavior of a functioning neural circuit are typically hidden from us. Minimally these include the membrane potential of every cell, but recordings of neural activity in behaving vertebrates such as mammals are limited to a small subset of cells. Even if all recorded cells reside in one circuit that we wish to describe, the circuit, which could be distributed or compact, receives inputs from tens of thousands of other neurons, whose activity is unknown. Moreover, even a continuous variable such as membrane potential is most commonly observed only at the discrete times of voltage spikes. Therefore, our descriptions of neural circuits require us to infer the behavior of underlying hidden variables when we observe only a sparse number of them.\n\nNumerous other properties impact the ongoing behavior of a cell and the circuit as a whole. These may include the number of vesicles of neurotransmitter and their voltage-dependent release probabilities at each synaptic connection or the cell-average states of activation and inactivation of the various ion channels. Calcium concentration and spatial distributions of all these values can also affect neural activity. Such an overwhelming abundance of known unknowns makes a full or complete description impossible and helps explain why we not only fail to have a concrete, detailed explanation of the behavior of most neural circuits but even do not know which dynamical system provides the best model of the behavior.\n\n\nClasses of dynamical system\n\nA dynamical system is any system that changes in time and can be described by a set of coupled differential equations. A pendulum is a simple example, the Hodgkin-Huxley model of a neuron a more complicated one, and the coordinated activity of all neurons in a brain an intractable one. To characterize a dynamical system rigorously, one should know how the rate of change of all relevant variables depends on the combination of their instantaneous values. One then can simulate how they change in time from any initial condition and plot these co-varying variables together as a trajectory. If a small change in initial conditions leads to identical behavior after some transient period, then the system possesses a point attractor state—trajectories converge if their initial difference is not too large. If the system is an oscillator, trajectories converge to a particular loop—a limit cycle—in which differences in initial conditions are maintained over time as a fixed phase offset. If trajectories diverge from each other across a broad range of initial conditions while all variables remain bounded, then the system is chaotic.\n\nHere we consider circuits with only two or three neurons to provide examples of many different types of dynamics. In general, a system with hundreds or thousands of neurons—so that we would need a space of hundreds or thousands of dimensions to plot the dynamics as the coordinated set of membrane potentials or firing rates of all neurons—could contain point attractors, limit cycles, and regions of chaos depending on which subsets of cells were more strongly active for one period of time. The richness of such high-dimensional systems and their relevance to cognitive function make them an important area of current study4–10.\n\n\nPoint attractors\n\nA point attractor state is equivalent to a stable fixed point of the dynamics, such as the bottom of a bowl with a ball in it. No neural circuit in vivo can strictly be in a point attractor state, as that would require all variables (such as membrane potentials) to be static. However, it may be reasonable to consider a broad average across variables, such as the mean firing rate of a large group of neurons, to be stationary following any initial transient response to a fixed input. Simple systems without feedback operate in such point attractor states if cells have one value of firing rate in the absence of stimulus and, typically following a period of adaptation, shift to a different stable firing rate in the presence of a stimulus (Figure 1). Neurons in the sensory periphery appear to behave in this manner.\n\n(A) Diagram of the model circuit. Arrows indicate excitatory connections, and balls indicate inhibitory connections between units. (B) Applied current as a function of time. Two different sized pulses of current are applied to unit 1. (C) Firing rate as a function of time in the coupled network. During each current step, a new attractor is produced, but following current offset the original activity state is reached. (D) Any particular combination of the firing rates of the two units (x-axis is rate of unit 1, y-axis is rate of unit 2) determines the way those firing rates change in time (arrows). Starting from any pair of firing rates, any trajectory following arrows terminates at the point of intersection of the two lines. Red line: nullcline for unit 2—the value of r(2) at which dr(2)/dt = 0 (its fixed point) given a value of r(1). Since unit 1 excites unit 2, the fixed point for r(2) increases with r(1). Black line: nullcline for unit 1—the value of r(1) at which dr(1)/dt = 0 (its fixed point) given a value of r(2). Since unit 2 inhibits neuron 1, the fixed point for r(1) decreases with r(2). The crossing point of the nullclines is a fixed point of the whole system. The fixed point is stable (so is an attractor state) because arrows converge on the fixed point. (E) As in (D) but the solution during the second pulse of applied current. The applied current shifts the nullcline for r(1) so that the fixed point of the system is at much higher values of r(1) and r(2). For parameters, see supporting Matlab code, “dynamics_two_units.m”.\n\nVariability in the spiking of a neuron—both within a trial and between trials—appears to be at odds with a point attractor description, which suggests a stable, stationary set of firing rates. However, such variability can be attributed to various noise terms that lead to each neuron’s spikes being produced randomly (say, as a Poisson process) with probability that depends on the rate—a hidden variable, which could be static and deterministic—and/or to noise-driven fluctuations in the rate about its stable fixed point. Thus, a point attractor framework is not incompatible with ever-varying neural activity, especially, as we shall discuss below, for systems with many attractor states.\n\nWhen a system possesses multiple point attractor states in the absence of stimuli, then the history of prior stimuli can determine the neural circuit’s current activity state—the particular attractor in which it resides—so the system can retain memories (Figure 2). In some of the most important pioneering work in computational and theoretical neuroscience11–14, Grossberg and Hopfield demonstrated how such discrete memory states can form via activity-dependent changes in the strength of connections between coactive neurons during stimulus presentation. While the initial analyses of the capacity (number of memories held) of such networks relied on binary neurons that were either on or off and updated in discrete time steps12, the principle of memory formation and memory retrieval via an imperfect stimulus has been demonstrated in continuous firing rate models in continuous time14,15 and in circuits of model spiking neurons16. These pattern-learning systems, known generically as autoassociative networks, provide great insight into how memories can be distributed across overlapping sets of cells and retrieved from imperfect stimuli via pattern completion to a point attractor state.\n\n(A) Diagram of the model circuit. Arrows indicate excitatory connections, and balls indicate inhibitory connections. The strong self-excitatory feedback renders each unit unstable once active. (B) Applied current as a function of time. The first pulse is applied to unit 1, the second to unit 2. (C) Firing rate as a function of time in the coupled network reveals three different activity states: both units inactive or either one active. The activity persists after offset of the applied current—a signature of multistability—so retains memory of past inputs. (D) Any particular combination of the firing rates of the two neurons (x-axis is rate of neuron 1, y-axis is rate of neuron 2) determines the way those firing rates change in time (arrows). Depending on the initial pair of firing rates, a trajectory following arrows terminates at one of the points of intersection of the two lines, either (0,0) or (100,0) or (0,100). The intersections at the midpoints of the lines are unstable—if activity of unit 1 is under 50 Hz, it decays to 0; if it is over 50 Hz, it will increase to 100 Hz (if unit 2 is inactive). Red line: nullcline for neuron 2—the value of r(2) at which dr(2)/dt = 0 (its fixed point) given a value of r(1). Since neuron 1 excites neuron 2, the fixed point for r(2) increases with r(1). Black line: nullcline for neuron 1—the value of r(1) at which dr(1)/dt = 0 (its fixed point) given a value of r(2). Since neuron 2 inhibits neuron 1, the fixed point for r(1) decreases with r(2). The crossing point of the nullclines is a fixed point of the whole system. The fixed point is stable (so is an attractor state) because arrows converge on the fixed point. (E) As in (D) but the solution during the second pulse of applied current. The applied current shifts the nullcline for r(1) so that the only fixed point of the system is at (100,0). For parameters, see supporting Matlab code, “dynamics_two_units.m”.\n\nThe value of a dynamical systems approach to understanding neural-circuit behavior that is otherwise highly counter-intuitive—even paradoxical—is exemplified by the inhibition-stabilized (IS) network. The behavior of the IS network is particularly worth taking the time to understand because there is evidence that regions of both the hippocampus and the cortex could operate in the IS regime.\n\nThe IS network is a feedback-dominated network in which self-excitation is strong enough to destabilize excitatory firing rates in the absence of feedback inhibition. However, feedback inhibition is also very strong, in fact dominant enough to clamp the excitatory firing at what is an otherwise unstable fixed point of the dynamics17. In IS circuits, the strong feedback inhibition is to similarly tuned neurons—to the same cells they receive excitation from—unlike most point attractor models in which the dominant inhibitory effect is cross-inhibition between differently tuned cells to enhance selectivity.\n\nAn intriguing property of the IS network is that a decrease in external excitatory input to inhibitory cells causes their steady-state firing rate to increase17. The initial transient decrease in firing of the inhibitory cells causes a strong increase in firing rate of the excitatory cells. The feedback loop is strong enough that the net effect on inhibitory cells, following the ensuing increase in their excitatory input, is an increase in firing rate (Figure 3). In the final state, with decreased external excitatory input to inhibitory cells, both inhibitory and excitatory cells have higher firing rate. The effect can be called paradoxical because in the final state the excitatory cells fire at a higher rate while receiving more inhibitory input than before.\n\nEvidence for neural circuit operation in the IS regime was first provided by a combination of modeling and data analysis for the hippocampus during theta oscillations, based on the relative phase relationship of oscillatory activity from excitatory and inhibitory cells17. More recently, strong evidence for operation in this regime has been provided for the primary visual cortex as an explanation of how two stimuli can switch from producing supralinear to sublinear summation as their contrast increases and the IS regime is reached18,19.\n\n(A) Diagram of the model circuit. Arrows indicate excitatory connections, and balls indicate inhibitory connections between units. Architecture is identical to that in Figure 1. (B) Applied current as a function of time. Two different sized inhibitory pulses of current are applied to unit 2. (C) Firing rate as a function of time in the coupled network. During each current step, a new attractor is produced, but following current offset the original activity state is reached. When inhibition is applied to unit 2, the rates of both unit 1 and unit 2 increase as a result of the ensuing net within-circuit increase in excitation to both units. The “paradox” lies in that external inhibition to unit 2 results in an increase in its rate (due to internal raised excitation) and, even more counter-intuitively, unit 1 stabilizes at a higher firing rate in the presence of greater inhibitory input from unit 2. (D) Any particular combination of the firing rates of the two units (x-axis is rate of unit 1, y-axis is rate of unit 2) determines the way those firing rates change in time (arrows). Starting from any pair of firing rates, any trajectory following arrows terminates at the point of intersection of the two lines. Red line: nullcline for unit 2—the value of r(2) at which dr(2)/dt = 0 (its fixed point) given a value of r(1). Since unit 1 excites unit 2, the fixed point for r(2) increases with r(1). Black line: nullcline for unit 1—the value of r(1) at which dr(1)/dt = 0 (its fixed point) given a value of r(2). Since unit 2 inhibits unit 1, at high enough r(2) only r(1) = 0 is possible and at low r(2) only r(1) = 100 is possible. The line joining the minimum and maximum values of r(1) would normally be of unstable fixed points, but in this system it is stabilized. The crossing point of the nullclines is the fixed point of the whole system. The fixed point is stable (so is an attractor state) because arrows converge on the fixed point. (E) As in (D) but the solution during the second pulse of applied current. The inhibitory applied current shifts the nullcline for r(2) down, and the result is that the fixed point of the system moves to higher values of both r(1) and r(2). For parameters, see supporting Matlab code, “dynamics_two_units.m”.\n\nThe term itinerancy is used if a system switches rapidly between distinguishable patterns of activity that last significantly longer than the switching time. Switches can occur by noise-driven fluctuations in a circuit with many stable point attractors20 or via biological processes such as synaptic depression or firing rate adaptation—which operate more slowly than changes in firing rates of cells—between quasistable attractor states21. A system with just two quasistable states can give rise to a relaxation oscillator by the latter process1,22,23, whereas a system with many quasistable states can give rise to “chaotic itinerancy”24,25.\n\nItinerancy through quasistable states can subserve sequence memory, with distinct states reached in response to both the number of stimuli and the types of stimuli in a sequence21. Noise-induced itinerancy through point attractor states can also serve as the neural basis of a sampling framework for Bayesian computation26 and can lead to optimal decision making if certain biological constraints must be met by the system27.\n\nPerhaps the most compelling evidence for attractor state itinerancy is during bistable percepts28–32, when the switching from one percept to the other and back arises in the presence of a constant, ambiguous stimulus such as the Necker cube33. Models of the phenomenon suggest a neural circuit possessing two attractor states with transitions between them produced by a combination of synaptic depression and noise-driven fluctuations34–36.\n\nIt is worth noting that experimental identification of such attractor state itinerancy may require non-standard analyses of neural spike trains. The standard practice of averaging across trials after their alignment to stimulus onset would fail to reveal such inherent dynamics because the timing of transitions varies across trials and indeed the initial state may differ from trial to trial. Thus, across-trial averaging would reveal simply a blurred, approximately constant rate dependent on the average response during the two distinct percepts. Therefore, it is essential to use single-trial methods of analysis where possible if one hopes to uncover such underlying dynamics. The use of averaging across trials has been a necessity when analyzing spike trains in vivo because of the apparent randomness and limited amount of information contained within the spike times of any one neuron in any one trial. However, methods such as hidden Markov modeling (HMM)37–39 allow one to treat each trial independently, especially if one has access to spike trains from many simultaneously recorded cells with correlated activity. HMM produces an analysis that assumes discrete states of activity with transitions between these activity states that vary in timing from trial to trial. Analysis of neural spike trains in vivo in multiple cortical areas by HMM and other methods has produced strong evidence for state transitions during cognitive processes, including motor preparation37,40, taste processing39, and perceptual decision making41,42.\n\nThe multistability necessary for memory is typically achieved in models via a subset of neurons switching from a low firing rate where the feedback activity is insufficient to generate a sustained response to a high firing rate state reinforced by recurrent excitatory feedback that is limited in rate only by saturation of an intrinsic or synaptic process. Unless the saturating process has a slow time constant, such active states maintained by recurrent feedback typically have much higher firing rates than those observed in vivo—indeed, in simple models, once excitatory feedback is increased enough to engender stable persistent activity in the absence of input, that activity can be at the neuron’s maximal firing rate, on the order of 100 Hz (as in Figure 2). Such rates are incompatible with the smaller changes of activity—often no more than 10 Hz between pre-stimulus and post-stimulus levels—during memory tasks in vivo43,44, calling into question the validity of these recurrent excitatory models.\n\nThis issue can be resolved by several different modeling assumptions45. If recurrent feedback current is mediated primarily through N-methyl-D-aspartate (NMDA) receptors and they are allowed a 100 ms time constant (though 50 ms may be more reasonable at in vivo temperatures for mammals), then the high firing rate state can be limited to 20 to 30 Hz46,47. Furthermore, if a slow time constant for synaptic facilitation of 7 seconds is used, then firing rates in the active state can be reduced to levels below 10 Hz48. A final possibility is that the network contains subgroups of excitatory and inhibitory cells operating in the IS regime (Figure 4) in which active subgroups can, in principle, maintain stable activity at arbitrary low rates while suppressing the activity of other subgroups via cross-inhibition49–51. In this regime, a system of point attractor states is compatible with the low firing rates of persistently active neurons observed in vivo.\n\n(A) Diagram of the model circuit. Arrows indicate excitatory connections, and balls indicate inhibitory connections between units. A third unit is added to the architecture of Figure 3. (B) Applied current as a function of time. The first current pulse is applied to unit 1, and the second is applied to unit 3. A small amount of noise is added to the current to prevent the system resting at an unstable symmetric state, toward which it is otherwise drawn. (C) Firing rate as a function of time in the coupled network. The current steps switch activity between stable states in which no neuron’s activity is greater than 10 Hz—note the difference in rates from the “traditional” bistability of Figure 2. (D) Any particular combination of the firing rates of the three units (x-axis is rate of unit 1, y-axis is rate of unit 3) determines the way those firing rates change in time (arrows). Only a plane out of the full three-dimensional space of arrows is shown—the plane corresponding to dr(3)/dt = 0. Starting from any pair of firing rates, any trajectory following arrows terminates at the point of intersection of the two lines. Red line: nullcline for unit 2—the value of r(2) at which dr(2)/dt = 0 (its fixed point) given a value of r(1). Black line: nullcline for unit 1—the value of r(1) at which dr(1)/dt = 0 (its fixed point) given a value of r(2). Since unit 2 inhibits neuron 1, the fixed point for r(1) decreases with r(2). The crossing points of the nullclines are fixed points of the whole system. The asymmetric fixed points are stable (so are attractor states) because arrows converge on them, whereas the intervening symmetric fixed point is unstable. For parameters, see supporting Matlab code, “dynamics_three_units.m”.\n\nA problem related to the one above is that most models of bistability produce spike patterns in the high-activity state that are much more regular than those observed in vivo. Systems that transition between various stable states will cause spike trains to be less regular because of the contribution of rate variation. Lower firing rates will likely also help with this problem because most model neurons produce irregular spike trains when operating below threshold in a fluctuation-driven regime at low firing rates. However, spike statistics such as the coefficient of variation (CV) of interspike intervals have not been analyzed to date in models of the IS regime.\n\n\nMarginal states (line attractors or continuous attractors)\n\nIf a dynamical system possesses a continuous range of points (a line) that variables of the system approach, then the attractor state has “marginal stability”; if the activity is perturbed away from the line, then it recovers toward the line, but deviations along the line can accumulate over time52. In neural circuit models, marginal states either depend on an underlying symmetry (for example, translation in space when considering memory for position via a ring attractor)53–56 or require other fine-tuning of parameters57–60. When neural activity enters a marginal state, the whole system can be described by a reduced number of variables, such as the position along the line of a line attractor61. Systems with marginal states are able to encode and store the values of continuous quantities53,54,60, integrate information over time perfectly55,57,62 (Figure 5), combine prior information with sensory input in a Bayesian manner63, and in general achieve optimal computational performance64. In practice, a system with many point attractor states that are close to each other—that is, total circuit activity differs little between states—can appear like a line attractor, performing integration8 yet with the benefit of greater robustness and stability65.\n\n(A) Diagram of the model circuit. Arrows indicate excitatory connections, and balls indicate inhibitory connections between units. The architecture is identical to that of Figure 2. (B) Applied current as a function of time. Two very small pulses of current are applied to unit 1. (C) Firing rate as a function of time in the coupled network. During each current step, the firing rate of unit 1 increases linearly because of the applied current. Inhibitory feedback to unit 2 causes a linear decrease in its firing rate. Upon stimulus offset, the firing rate reached is maintained. (D) Any particular combination of the firing rates of the two units (x-axis is rate of unit 1, y-axis is rate of unit 2) determines the way those firing rates change in time (arrows). Starting from any pair of firing rates, any trajectory following arrows terminates at a point where the two lines overlap each other. Red line: nullcline for unit 2—the value of r(2) at which dr(2)/dt = 0 (its fixed point) given a value of r(1). Black line: nullcline for unit 1—the value of r(1) at which dr(1)/dt = 0 (its fixed point) given a value of r(2). Since the two units inhibit each other, the overlapping lines have negative gradient. (E) A small applied current to unit 1 shifts its nullcline to the right slightly. Now the only fixed point is where the two lines intersect at r(2) = 0, but in the region between the two parallel nullclines, the rate of change is small (note the small arrows parallel to the lines) so firing rates change gradually. For parameters, see supporting Matlab code, “dynamics_two_units.m”.\n\nPredicted experimental signatures of marginal states include drift of neural activity as noise accumulates in the manner of a random walk—leading to variance increasing linearly with time54—in the presence of a constant stimulus; perfect temporal integration of inputs55,57; and correlations within single neural spike trains that decay linearly over time52,66. These have acquired some degree of experimental support67–69.\n\n\nOscillating systems (cyclic attractors or limit cycles)\n\nObservations of oscillations are widespread throughout the brain70,71—indeed, the earliest human extracranial recordings revealed oscillations in electrical potential72. In some cases, the presence of oscillations is inferred from a peak in the power spectrum at a particular frequency73,74—such a peak could arise from attractor state itinerancy, chaos, or heteroclinic orbits (see below) in the absence of a true oscillator. Yet, given the overwhelming abundance of evidence, there is no doubt as to the existence of oscillations in neural circuits—something still in question for other dynamical frameworks discussed here. Therefore, current research focuses on elucidating the role, if any, of oscillations in diverse mental processes75,76.\n\nSpiking neurons themselves can be oscillators, so it should not seem surprising that neural circuits can also oscillate. Circuit oscillations can arise from the intrinsic oscillations of constituent neurons, or from the circuit connectivity (Figure 6), or from a combination of the two. In general, any system with fast positive feedback and slower negative feedback is liable to oscillate (Figure 7). Moreover, in any nonlinear dynamical system, a change in the inputs leads to a change in amplitude and frequency of ongoing oscillations, so correlations between oscillatory power or frequency and task condition are inevitable. Therefore, observed correlations between oscillatory power or synchrony and behavior or cognitive process—whether related to attention77, arousal78, memory load79, or sleep state80–82—may indicate a causal dependence in one direction (oscillations cause the process) or the other (particular processes cause oscillations as an epiphenomenon). The difficulty of distinguishing the two arises because experiments aimed at altering an oscillation inevitably alter other properties of the dynamical system necessary for the cognitive process.\n\n(A) Diagram of the model circuit. Arrows indicate excitatory connections, and balls indicate inhibitory connections between units. The architecture is identical to that of Figure 1 and Figure 3. (B) Applied current as a function of time. An inhibitory pulse of current is applied to unit 1. (C) Firing rate as a function of time in the coupled network. Oscillations are switched off by the inhibition to unit 1, so the system has two stable attractors: one a limit cycle, the other a point attractor. (D) Any particular combination of the firing rates of the two units (x-axis is rate of unit 1, y-axis is rate of unit 2) determines the way those firing rates change in time (arrows). Depending on the starting point, a trajectory following the arrows will fall on the limit cycle (the green closed orbit, which represents the coordinated variation of firing rate with time during the oscillations) or will reach the fixed point at the origin. Red line: nullcline for unit 2—the value of r(2) at which dr(2)/dt = 0 (its fixed point) given a value of r(1). Since unit 1 excites unit 2, the fixed point for r(2) increases with r(1). Black line: nullcline for unit 1—the value of r(1) at which dr(1)/dt = 0 (its fixed point) given a value of r(2). Crossing points of the nullclines are the fixed points of the whole system, but the one within the limit cycle is unstable, as is the one with r(2) = 0 but r(1) > 0. (E) As in (D) but the solution during the inhibitory pulse of applied current. The inhibitory current shifts the nullcline for r(1) down, and the result is that all trajectories terminate at the origin. For parameters, see supporting Matlab code, “dynamics_two_units.m”.\n\n(A) Diagram of the model circuit. Arrows indicate excitatory connections, and balls indicate inhibitory connections between units. Architecture is identical to that of Figure 4. (B) Applied current as a function of time. The first current pulse is applied to unit 3, and the second is applied to unit 1. (C) Firing rate as a function of time in the coupled network. The current steps switch activity between two stable states with different frequencies of oscillation. (D) Any particular combination of the firing rates of the three units (x-axis is rate of unit 1, y-axis is rate of unit 3) determines the way those firing rates change in time (arrows). Only a plane out of the full three-dimensional space of arrows is shown: the plane corresponding to dr(3)/dt = 0. Red line: nullcline for unit 2—the value of r(2) at which dr(2)/dt = 0 (its fixed point) given a value of r(1). Black line: nullcline for unit 1—the value of r(1) at which dr(1)/dt = 0 (its fixed point) given a value of r(2). The crossing points of the nullclines are fixed points of the whole system, none of which is stable in this example. For parameters, see supporting Matlab code, “dynamics_three_units.m”.\n\nThe importance of oscillations is perhaps undisputed only in the case of motor systems that produce a repeating, periodic output83 or in the sensations of whisking and olfaction that are directly related to such motor rhythms. Perhaps the most accepted roles for information processing by oscillations are within hippocampal place cells, whose phase of firing with respect to the ongoing 7 to 10 Hz theta oscillation contains substantial information84–87.\n\nOscillating systems contain limit cycles and so appear as closed loops in a plot of one variable against another (Figure 6C). Since activity is attracted to the limit cycle—which is a line embedded within a higher-dimensional space—oscillating systems have some similarities to line attractors. In particular, small perturbations can be accumulated in the phase of the oscillation (along the line of the limit cycle), so, as with line attractors, noise accumulates as a random walk in one particular direction. Moreover, the phase of an oscillator retains a memory of perturbations, so oscillators can also be integrators, albeit only up to an offset of one cycle and with the need of an unperturbed oscillator for comparison.\n\n\nChaotic systems (strange attractors)\n\nA high-dimensional neural system—as arises if the activity of each neuron can vary with little correlation with the activity of other neurons—with a balance between excitatory and inhibitory random connections becomes chaotic if connections are strong enough88–90. An early model of eye-blink conditioning in the cerebellum (the part of the mammalian brain with the highest density of cells and connections) used a chaotic circuit to encode and reproduce timing information91,92.\n\nThe smallest change in the initial conditions of a chaotic system leads to an indeterminate change in response, which can pose a serious problem for information processing and memory (Figure 8). Yet a system operating near or at the “edge of chaos” can be computationally efficient93 and become reliably entrained to inputs while responding more rapidly than ordered systems89,94. Moreover, certain learning rules for changing of connection strengths between neurons in a chaotic system can allow the encoding of almost any spatiotemporal input pattern4,95, the switching between multiple patterns4, and the encoding and processing of many rule-based tasks6,8. Thus, chaotic systems appear to be highly flexible and trainable.\n\n(A) Diagram of the model circuit. Arrows indicate excitatory connections, and balls indicate inhibitory connections between units. Architecture is identical to that of Figure 4 and Figure 7. (B, C) Firing rate as a function of time in the coupled network, from two imperceptibly different initial conditions. No applied current is present. The miniscule difference in initial conditions is amplified over time, so, for example, unit 3 produces a small burst of activity after 1.5 s in (D), but that burst is absent from (C). (D) Any particular combination of the firing rates of the three units (x-axis is rate of unit 1, y-axis is rate of unit 3) determines the way those firing rates change in time (arrows). Only a plane out of the full three-dimensional space of arrows is shown: the plane corresponding to dr(2)/dt = 0. Red line: nullcline for unit 2—the value of r(2) at which dr(2)/dt = 0 (its fixed point) given a value of r(1). Black line: nullcline for unit 1—the value of r(1) at which dr(1)/dt = 0 (its fixed point) given a value of r(2). The crossing points of the nullclines are unstable fixed points of the whole system. For parameters, see supporting Matlab code, “dynamics_three_units.m”.\n\nObserved signatures of chaos include the apparent randomness and variability of spike trains, especially during spontaneous activity in the absence of stimuli, and the initial drop of such variability upon stimulus presentation94,96.\n\nChaos can arise in systems with quasistable attractor states as an itinerancy between the states in an order possessing no pattern97,98 or in an oscillating system (typically as unpredictable jumps between different types of oscillation). Heteroclinic sequences (see below) can also be chaotic99. Addition of a small amount of intrinsic noise to chaotic systems causes the divergence in activity observed without noise upon small changes in initial conditions to occur on separate trials with identical initial conditions.\n\n\nHeteroclinics\n\nA common type of fixed point in a system with many variables (meaning it is high-dimensional) is a saddle point. Saddle points are so-named because, like the saddle on a horse or the saddle on a ridge, there are directions where the natural tendency is to approach the fixed point (moving down from a higher point on the ridge) and other directions where the natural tendency is to move away from the fixed point (down to the valley below). If the state of a dynamical system can move toward one such saddle point and then move away from it to another one, and so on, the trajectory is called a heteroclinic sequence.\n\nHeteroclinic sequences have similarities to systems with attractor-state itinerancy and a type of oscillator that switches between states that appear stable on a short timescale, called a relaxation oscillator. All three systems have states toward which the system is drawn but at which the system does not remain. In a heteroclinic sequence, activity can be funneled toward each saddle point as if it were an attractor state but, once in the vicinity of the saddle point, will find the direction of instability and move away (Figure 9). In the absence of noise, the duration in a “state” (the vicinity of a particular saddle point) depends on how close to the fixed point the system gets and therefore may vary with initial conditions. Interestingly, a small amount of noise can make these state durations more regular100.\n\n(A) Diagram of the model circuit. Arrows indicate excitatory connections, and balls indicate inhibitory connections between units. (B, C) Firing rate as a function of time in the coupled network from different initial conditions (near the fixed points) with no applied current. Activity is initially slow to move away from the vicinity of the fixed point (along its unstable direction) but after a cycle returns to the vicinity of the same fixed point (along its stable direction). (D) Any particular combination of the firing rates of the three units (x-axis is rate of unit 1, y-axis is rate of unit 3) determines the way those firing rates change in time (arrows). Only a plane out of the full three-dimensional space of arrows is shown: the plane corresponding to dr(2)/dt = 0. Red line: nullcline for unit 2—the value of r(2) at which dr(2)/dt = 0 (its fixed point) given a value of r(1). Black line: nullcline for unit 1—the value of r(1) at which dr(1)/dt = 0 (its fixed point) given a value of r(2). The crossing points of the nullclines are fixed points of the whole system, in this case at r(1) = 100, r(2) = 0, and r(3) = 0 and at r(1) = 0, r(2) = 100, and r(3) = 100. The fixed points are saddle points in that firing rates can either approach the fixed point or move away from it, depending on the precise set of rates. For parameters, see supporting Matlab code, “dynamics_three_units.m”.\n\nModels of heteroclinic sequences have been proposed as a basis for memory101,102 and decision making103,104. According to some calculations, a randomly connected neural circuit would contain a suitable number of heteroclinic trajectories for information processing105. In the high-dimensional space of neural activity, most random fixed points would have some directions along which they attract neural activity and other directions along which they repel it, meaning that they would be the saddle points necessary for producing heteroclinic sequences. However, no unique predictions of cognitive processing via heteroclinic sequences have been linked to empirical data to date.\n\n\nCriticality\n\nMany scientists argue that the brain is in a critical state, in fact exhibiting self-organized criticality, so should be studied as such. Criticality is a measured state of a system rather than a dynamical model. A critical system is characterized by a number of properties, including the following: power law decays of the durations and sizes of features such as neural avalanches106; relationships between the exponents of these different power laws; a scaling of the time dependence of these features when binned by size onto a universal curve107; and correlations of fluctuations that extend across the system. All of these features arise from a particular distribution of the number of possible states binned according to their probability of occurrence—a distribution that, in principle, can be enumerated108—that conspire to remove any stereotypical size of the systems fluctuations. Critical systems have been argued to be optimal at information processing93,109.\n\nIt is likely that most of the above dynamical systems under appropriate conditions could produce critical behavior110—although the term criticality is sometimes used for systems operating at the edge of chaos93 where critical properties can arise in random networks111. Criticality appears to be more strongly dependent on the modular or hierarchical structure of network connectivity112–114 than the within-module dynamics.\n\nCharacteristics of criticality have been measured in cortical cultures106,107, intact retina108, and whole-brain imaging115. Observations to date suggest that much neural activity is close to being critical109,116 rather than exactly critical, leaving it open that even if a particular dynamical system cannot engender exact criticality, it is still close enough to provide an accurate description of neural activity (but see 108).\n\n\nSummary\n\nMany different types of dynamical system have been proposed as models of neural activity, each of which can be justified by experimental evidence in some brain regions and circumstances. Different neural circuits in different parts of the brain may operate in different dynamical regimes because of connectivity differences, in particular differences in relative dominance of feedback or feedforward connections and in the relative contributions of excitatory or inhibitory neurons. Moreover, since the dynamical regime of a neural circuit depends on many factors (only one of which is the connectivity pattern), it is likely that a single circuit can change between dynamical regimes following learning or when it receives inputs or neural modulation.\n\nAny one of these types of dynamical system (or any other not mentioned) could provide the most accurate basis for understanding a particular neurological function, another may be compatible with the observed data and more useful for explaining particular features of the neural behavior, while others may at heart be incompatible with the known circuit properties. Therefore, it behooves us all to avoid entrenchment in our favorite paradigm and to improve our understanding of the variety of dynamical systems when attempting to understand neural circuit function.\n\nA final point worth making is that just knowing the connectome—which neurons are connected to each other—does not tell us about the operation of a neural circuit. Many of the dynamics we see arise in the same simple circuit with the same neurons (for example, Figure 1, Figure 3, and Figure 6 have identical architecture, even accounting for type of synapse, as do Figure 4, Figure 7, and Figure 8); differences in neural excitability or differences in strengths of connections can produce different functionality within a single architecture. Conversely, distinct connectivity patterns can give rise to the same function. Rather, observation of the coordinated activity of many neurons during mental processing is the route to understanding the remarkable abilities of our brains.",
"appendix": "Competing interests\n\n\n\nThe author declares that he has no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\ndynamics_two_units.\n\nClick here to access the data.\n\ndynamics_three_units.\n\nClick here to access the data.\n\n\nReferences\n\nIzhikevich EM: Solving the distal reward problem through linkage of STDP and dopamine signaling. Cereb Cortex. 2007; 17(10): 2443–52. PubMed Abstract | Publisher Full Text\n\nStrogatz SH: Nonlinear Dynamics and Chaos. Boulder, CO: Westview Press, 2015. Reference Source\n\nDayan P, Abbott LF: Theoretical Neuroscience. MIT Press, 2001. Reference Source\n\nSussillo D, Abbott LF: Generating coherent patterns of activity from chaotic neural networks. Neuron. 2009; 63(4): 544–57. 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PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "13417",
"date": "24 May 2016",
"name": "John Beggs",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13416",
"date": "24 May 2016",
"name": "Mark Goldman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13418",
"date": "24 May 2016",
"name": "Harel Shouval",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-992
|
https://f1000research.com/articles/5-990/v1
|
24 May 16
|
{
"type": "Review",
"title": "Cannabinoid receptor type-1: breaking the dogmas",
"authors": [
"Arnau Busquets Garcia",
"Edgar Soria-Gomez",
"Luigi Bellocchio",
"Giovanni Marsicano",
"Edgar Soria-Gomez",
"Luigi Bellocchio",
"Giovanni Marsicano"
],
"abstract": "The endocannabinoid system (ECS) is abundantly expressed in the brain. This system regulates a plethora of physiological functions and is composed of cannabinoid receptors, their endogenous ligands (endocannabinoids), and the enzymes involved in the metabolism of endocannabinoids. In this review, we highlight the new advances in cannabinoid signaling, focusing on a key component of the ECS, the type-1 cannabinoid receptor (CB1). In recent years, the development of new imaging and molecular tools has demonstrated that this receptor can be distributed in many cell types (e.g., neuronal or glial cells) and intracellular compartments (e.g., mitochondria). Interestingly, cellular and molecular effects are differentially mediated by CB1 receptors according to their specific localization (e.g., glutamatergic or GABAergic neurons). Moreover, this receptor is expressed in the periphery, where it can modulate periphery-brain connections. Finally, the better understanding of the CB1 receptor structure led researchers to propose interesting and new allosteric modulators. Thus, the advances and the new directions of the CB1 receptor field will provide new insights and better approaches to profit from its interesting therapeutic profile.",
"keywords": [
"Endocannabinoid system",
"allosteric modulator",
"molecular pharmacology",
"cannabinoid ligands",
"CB1 receptor signaling"
],
"content": "Introduction\n\nThe endocannabinoid system (ECS) is composed of G protein-coupled cannabinoid receptors, namely cannabinoid receptor-1 (CB1) and cannabinoid receptor-2 (CB2)1,2; the endogenous cannabinoids called endocannabinoids, such as the lipids anandamide and 2-arachidonoylglycerol3,4; and the enzymes involved in their synthesis and inactivation5. The family of endocannabinoids has recently grown to include a group of peptide ligands (so-called pepcans) and other lipid molecules, such as lipoxin and pregnenolone, interestingly acting as allosteric enhancers or signal-specific inhibitors (SSIs) of CB1 receptors6.\n\nOne of the main characteristics of the ECS is its broad distribution throughout the body. In this review, we will specifically focus our attention on the CB1 receptor-dependent functions in the nervous system (particularly the brain). The CB1 receptor is considered the most abundant metabotropic receptor in the brain7. It was cloned in 19901 and its distribution has been well characterized in both rodents8,9 and humans10. These receptors are particularly rich in the central nervous system11,12, where they control a wide spectrum of physiological and pathological conditions, including brain development, learning and memory, motor behavior, regulation of appetite, body temperature, pain perception, inflammation, and they are involved in various psychiatric, neurological, and neurodevelopmental disorders13–17.\n\nThis review highlights recent findings that challenge or extend accepted “dogmas” of CB1 receptor signaling. Thus, it discusses where CB1 receptors are localized, the importance of CB1 receptors outside the brain, and new strategies to pharmacologically act on these receptors. Importantly, the understanding of where, which, and how CB1 receptor function is mandatory to improve the pharmacological strategies to act on this promising therapeutic target.\n\n\nLocalization of CB1 receptors in different neuronal types\n\nCB1 receptor localization has been widely studied during the last few decades18. Thus, early studies provided strong evidence for a presynaptic localization of CB1 receptors, from where they can control the neurotransmitter release7,19. However, the somatodendritic localization of CB1 receptors cannot be discarded, as processes of self-inhibition through these receptors have been demonstrated in the cortex20–23. According to this, recent work describes that somatodendritic CB1 receptors control a specific postsynaptic signaling cascade important for the cognitive impairment induced by cannabinoids24. Therefore, more studies are needed to clarify the relative involvement of pre- or post-synaptic CB1 receptors in brain functions and how this can affect our general view of how the ECS controls synaptic transmission.\n\nInterestingly, new experimental approaches (e.g., imaging tools) have shown the expression of CB1 receptors in different neuronal types, including GABAergic, glutamatergic, and serotonergic neurons, among others8,25–28. Moreover, although the anatomical presence of CB1 receptors in cholinergic, noradrenergic, or dopaminergic neurons has not been fully characterized, cannabinoids are known to control acetylcholine and dopamine release29,30. For example, it has been recently shown that CB1 receptors can specifically control cholinergic over glutamatergic transmission at single synapses that co-release both neurotransmitters31.\n\nImportantly, the expression levels of CB1 receptors can drastically differ among different cell types and can diverge between different brain regions12,25,32,33. This widely distributed and differential expression in the brain reflects the complexity, and can explain the variety of functions, of the ECS. For instance, this specific distribution can explain some of the bimodal effects of cannabinoid drugs34,35. Thus, recent studies demonstrated how CB1 receptors localized in GABAergic neurons can control food intake34, running related behaviors36,37, drug addiction38,39, and learning and memory processes40,41, among other behaviors, whereas CB1 receptors localized in glutamatergic neurons control neuroprotection42, olfactory processes25, fear memories43, social behaviors44, and anxiety35, among others. Moreover, CB1 receptors present in serotonergic neurons can modulate emotional responses45.\n\n\nLocalization of CB1 receptors in other cell types or intracellular organelles\n\nThe biased neuron-centric view in the ECS field changed when CB1 receptors were found in another type of brain cells, the glial cells46–49. Moreover, recent studies have demonstrated how the astroglial CB1 receptor can modulate important physiological functions in behavior and synaptic plasticity such as learning and memory and long-term depression in the hippocampus50–52. Therefore, this receptor can shape synaptic transmission via astroglial signaling53. By doing this, it modulates the effects of exogenous cannabinoids on working memory46 and, notably, can also determine the selective activity of specific circuits in the striatum54. Thus, the improvement of the current tools will consolidate this knowledge to better elucidate the role of CB1 receptors and astrocytes on brain functioning55. Interestingly, recent findings have shown how CB1 receptors can modulate microglia activation, suggesting its presence in this cell type49.\n\nAlthough CB1 receptors are localized primarily at the plasma membrane, more and more evidence suggests the presence of functional intracellular CB1 receptors56,57. For instance, a portion of these receptors is functionally present in cell mitochondria58. In the past, previous data showed that cannabinoids can alter mitochondrial functions, but these effects were fully ascribed to unspecific membrane disturbance induced by these lipid molecules59,60. However, recent results challenge this idea, indicating that CB1 receptors are also present in mitochondrial membranes in the periphery, such as in spermatozoa61 or skeletal muscles62, and in the brain, where they directly regulate mitochondrial oxidative phosphorylation (OXPHOS) activity58,63,64 or can impact feeding behavior65. However, further studies and more direct, specific, and powerful tools are needed to investigate the role of mitochondrial or other intracellular CB1 receptors on synaptic transmission, brain functions, and behavior. Interestingly, brain mitochondrial functions have been recently causally associated to anxiety-related responses in the nucleus accumbens66, demonstrating how brain energetics can impact behavior.\n\n\nLocalization of CB1 receptors in the periphery\n\nIn the last two decades, CB1 receptors have been described in a number of peripheral tissues, including fat tissue67, gastrointestinal tract68, mouth and oral cavity69, eye70, cardiovascular system71, liver72, pancreas73, immune system74, bone75, skin76, and skeletal muscle77. Indeed, it seems that the ECS is present in a large majority of tissues and its specific functions have recently been investigated78.\n\nThe complex interactions between peripheral organs and the central nervous system raised a particular interest within the neuroscience field. In this sense, it is worth discussing how the peripheral processes modulated by the CB1 receptors are affecting the central nervous system functions. A recent study demonstrated that the peripheral sympathetic activity controlled by CB1 receptors is necessary for central functions, such as hypophagia and anxiety-like effects79. Other potential examples of the roles of CB1 receptors in the periphery-brain connection are the control of the release of stress hormones from the adrenal glands80 or the modulation of gut functions impacting on behavioral responses. Indeed, a close interaction between adipose tissue, gut bacteria, and the endocannabinoid system has been proposed in the context of obesity81,82.\n\n\nNew advances in the CB1 receptor pharmacology\n\nSeveral orthosteric ligands of CB1 receptors have been described in the last few decades, including natural or synthetic CB1 receptor agonists (e.g., Δ9-tetrahydrocannabinol [THC], CP-55,940), antagonists (e.g., rimonabant), and orthosteric endocannabinoids6,83. Moreover, endocannabinoids seem also to target non-cannabinoid receptors (e.g., G protein-coupled receptor 55 receptors)84,85 and ion channels (e.g., serotonergic, nicotinic acetylcholine receptors, or vanilloid receptors)86, particularly at concentrations at which they have been found to interact with CB1 or CB2 receptors6,87. Notably, the orthosteric action of CB1 receptor agonists and antagonists induces important side effects88,89. For example, rimonabant, known as a partial antagonist/inverse agonist, showed different side effects in humans88. In this sense, different strategies have been shown to improve the safety profile and overcome the side effects induced by CB1 antagonists, such as the neutral CB1 antagonists90.\n\nInterestingly, the pharmacology of CB1 receptors is nowadays also focused in the recent developments on putative allosteric binding sites of these receptors and how this can be translated into new therapeutic approaches. As cannabinoid ligands present an interesting therapeutic profile91, the development of new and safer drugs such as CB1 receptor allosteric modulators is needed. Indeed, this strategy has become a hot topic in the G protein-coupled receptors field and there are different positive and negative allosteric modulators described (PAMs and NAMs, respectively)92,93. Consequently, different compounds have been developed as exogenous CB1 allosteric modulators, including the indole derivatives (e.g., the NAM “ORG” compounds)94, urea derivatives (e.g., the NAM PSNCBAM-1)95, and other small molecules that also display a PAM profile, such as RTI-37196. Importantly, recent work also identified natural PAMs and NAMs of CB1 receptors, such as the lipoxin A4, the hemopressin pepcan-12, and pregnenolone97,98, which might represent model chemical structures for the development of new drugs. Although numerous studies have fully characterized the chemical and signaling properties of these new synthetic or natural compounds97,98, the in vivo effects of all these drugs modulating physiological or pathological conditions constitutes an emerging area in the cannabinoid field. In this context, the neurosteroid pregnenolone exerts peculiar effects on CB1 receptor signaling. Indeed, pregnenolone, by binding to a specific identified site on CB1 receptors, displays an interesting SSI profile: whereas CB1-dependent modulation of cytoplasmic cyclic AMP signaling is unaltered by pregnenolone, the neurosteroid fully blocks the activation of extracellularly regulated kinases (ERKs) and the inhibition of mitochondrial activity by cannabinoids63. By these mechanisms, the SSI pregnenolone blocks different central effects of THC, including memory impairment, hypolocomotion, and cannabinoid self-administration in rodents63. Other compounds have been shown to alter CB1 receptor-dependent effects. For instance, the synthetic PAM ZCZ011 reduces neuropathic pain99, whereas the PAM lipoxin A4 shows anti-inflammatory effects100. Interestingly, it was recently shown that cannabidiol, which has been previously reported as a CB1 receptor antagonist, behaves also as a non-competitive NAM of CB1 receptors, despite its low affinity to these receptors101.\n\nThe allosteric modulators of CB1 receptors are not the only therapeutic agents recently proposed. Indeed, the effects of several phytocannabinoids in preclinical models of central nervous system diseases and, where available, clinical trials have been investigated, suggesting a promising phytocannabinoid-based medicine102. Another factor that can change the CB1 receptor pharmacology is heteromerization with other receptors. Heteromers of CB1 receptors and other proteins recently emerged as an important target of the in vivo effects of cannabinoids103–105. Notably, these heterocomplexes could be potentially modulated104 and this implies another pharmacological tool to act on CB1 receptor signaling. Moreover, present evidence points to the membrane environment as another critical regulator of CB1 receptor signaling, and this can be potentially exploited for the development of novel therapeutic compounds106. Finally, a G protein-coupled receptor such as the CB1 receptor may also have a constitutive, ligand-free mode of signaling, as has been shown in hippocampal GABAergic synapses107. All of these new ideas demonstrate that the research community may dedicate more effort to tackle CB1 receptors.\n\n\nConclusions\n\nThis short review focused on the new findings in CB1 receptor research. However, the ECS comprises other components such as CB2 receptors, the endocannabinoids, and the enzymes responsible for their synthesis and degradation. In this sense, recent advances have demonstrated the importance of CB2 receptors in the brain108–110, the presence of other endocannabinoid-like molecules111,112, other potential receptors that can be activated by endocannabinoids87, and interesting findings regarding the localization and pharmacology of the enzymes involved in the metabolism of these endocannabinoids113,114. In brief, the actual picture of how the endocannabinoid system works is quite complicated and more efforts are needed to try to merge the old and the new ideas in this field (Figure 1).\n\nOn the left panel, the classic view of the CB1 receptor is represented. The CB1 receptor was thought to be exclusively localized in GABAergic neurons, where it was demonstrated to inhibit neurotransmitter release. On the right panel, the current view of the CB1 receptor is illustrated. Different advances have completely changed this picture: (A) The CB1 receptor is present in different neuronal types and in glial cells, both in astrocytes (B) and potentially in microglia (C). Furthermore, it is found intracellularly in the mitochondria (D) and endosomes (E). The view of a canonical retrograde system changed after the CB1 receptor localization in postsynaptic somatodentritic neurons was demonstrated (F). Nowadays, we know that CB1 receptor presents allosteric binding sites (G) and that it could form heteromers (H). Beyond the brain, the CB1 receptor is widely expressed in the periphery (I), where it can modulate the periphery-brain connection. All of this new knowledge reflects the complexity of the central nervous system and the advance in neuroscience, positing the CB1 receptor as an ideal tool for studying brain functions. CB1, cannabinoid receptor-1; CB2, cannabinoid receptor-2; eCB, endocannabinoid; NT, neurotransmitter.\n\nAn open question in the cannabinoid field is whether the cellular diversity of CB1 functions could improve the therapeutic exploitation of cannabinoid-based drugs. One can speculate whether different CB1 ligands can mediate different signaling pathways by selectively controlling different CB1 receptors present in different cellular populations. Likewise, it is possible that specific drugs could target exclusively mitochondrial CB1 (mtCB1) receptors or could avoid activation of intracellular pools of CB1. More studies will be needed to answer these questions, but there is already some evidence demonstrating a different pharmacological profile between CB1 receptors expressed in GABAergic and glutamatergic cells. Thus, “glutamatergic” CB1 receptors are more sensitive to low doses of agonists and are endowed with stronger intracellular coupling, whereas “GABAergic” pools of the receptor are activated by higher doses of agonists and produce lower activation of G proteins34,35,43,115. Therefore, one could speculate that specific compounds able to selectively activate different cellular subpopulations of CB1 receptors could be developed. Moreover, combinations of drugs able to modulate glutamatergic or GABAergic neurotransmission with cannabinoid agonists have been shown to promote specific effects of CB1 receptors and inhibit others116. It is also interesting to note that both perisomatic and dendritic GABAergic synapses use phasic endocannabinoid signaling, but the tonic form of cannabinoid signaling is present only in perisomatic cells107. Moreover, a recent study80 shows that the peptide endocannabinoids, known as pepcans, act as endogenous allosteric modulators of CB1 activity exclusively on noradrenergic neurons, demonstrating a cell type-specific regulatory role on endocannabinoid signaling. All of these new and exciting findings suggest that the better we understand cannabinoid signaling, the closer we are to developing specific and local pharmacological drugs that may have importance in brain disorders.\n\nOverall, the new and exciting findings suggesting different and specific localizations of the ECS components and the new strategies proposed to tackle their activity of this receptor open the door to new questions (Table 1). Indeed, the endocannabinoid system has been related to many physiological and pathological functions13,18,117, and the better understanding of these new evidences will bring more light to exploit the therapeutically beneficial properties of this widely spread neuromodulator system in the brain and in the body.\n\nCB1, cannabinoid receptor-1; CB2, cannabinoid receptor-2.\n\n\nAbbreviations\n\nCB1, cannabinoid receptor-1; CB2, cannabinoid receptor-2; ECS, endocannabinoid system; NAM, negative allosteric modulator; PAM, positive allosteric modulator; SSI, signal-specific inhibitor; THC, Δ9-tetrahydrocannabinol.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nWe thank all the members of the GM lab for useful discussions. This work was supported by INSERM (to GM), EU-FP7 (PAINCAGE, HEALTH-603191 to GM and FP7-PEOPLE-2013-IEF-623638 to AB-G), European Research Council (Endofood, ERC-2010-StG-260515; CannaPreg, ERC-2014-PoC-640923, to GM), Fondation pour la Recherche Medicale (DRM20101220445, to GM and LB), Human Frontiers Science Program (to GM), Region Aquitaine (to GM), French State/Agence Nationale de la Recherche/LabEx BRAIN (ANR-10-LABX-0043 to GM), Fyssen Foundation (to ES-G), CONACyT (to ES-G), French State/Agence Nationale de la Recherche/IdEx (ANR-10-IDEX-03-02 to AB-G), and French State/Agence Nationale de la Recherche/Blanc (NeuroNutriSens ANR-13-BSV4-0006-02 to GM).\n\n\nReferences\n\nMatsuda LA, Lolait SJ, Brownstein MJ, et al.: Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature. 1990; 346(6284): 561–4. PubMed Abstract | Publisher Full Text\n\nHowlett AC, Barth F, Bonner TI, et al.: International Union of Pharmacology. XXVII. 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{
"id": "13724",
"date": "24 May 2016",
"name": "Alberto Bacci",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13723",
"date": "24 May 2016",
"name": "Istvan Katona",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13722",
"date": "24 May 2016",
"name": "Carsten T Wotjak",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-990
|
https://f1000research.com/articles/5-986/v1
|
24 May 16
|
{
"type": "Review",
"title": "Biomarkers of acute kidney injury and associations with short- and long-term outcomes",
"authors": [
"Jennifer A. Schaub",
"Chirag R. Parikh",
"Jennifer A. Schaub"
],
"abstract": "Acute kidney injury is strongly associated with increased mortality and other adverse outcomes. Medical researchers have intensively investigated novel biomarkers to predict short- and long-term outcomes of acute kidney injury in many patient care settings, such as cardiac surgery, intensive care units, heart failure, and transplant. Future research should focus on leveraging this relationship to improve enrollment for clinical trials of acute kidney injury.",
"keywords": [
"acute kidney injury",
"AKI",
"biomarkers",
"chronic kidney disease",
"CKD"
],
"content": "Introduction\n\nAcute kidney injury (AKI) is a common complication, occurring in about 5% of hospitalizations, and is often associated with various short- and long-term complications1. AKI has been associated with short-term outcomes, such as increased length of hospital stay, length of ventilation, and in-hospital mortality2. A meta-analysis of observational studies demonstrated that survivors of AKI had a long-term mortality rate of more than twice that of patients without AKI3. Furthermore, increasing duration of AKI was positively associated with long-term mortality4,5. Although both the magnitude and length of AKI are significantly associated with reduced long-term survival, no clear causal relationship between AKI and mortality has been established yet. However, mechanisms are currently being elucidated, and there is increasing evidence that AKI can cause distant organ injury, including lung and cardiac injury6. Despite the relationship between AKI and adverse clinical outcomes, clinical trials investigating novel treatments for AKI have not demonstrated any benefit7–11.\n\nWhile increased mortality is a well-documented complication of AKI, evidence from basic science and clinical literature suggests that there may be other consequences, including increased risk for development of hypertension, increased risk of progression to chronic kidney disease (CKD), and increased risk of cardiovascular events. Survivors of AKI are about 20 percent more likely to develop elevated blood pressure after an AKI event12. Rat models have shown that there is rarefaction of peritubular capillaries after an episode of AKI, which leads to salt-sensitive hypertension13,14. A meta-analysis of observational data demonstrates that AKI increases the risk of CKD by almost eightfold15. Cell cycle arrest in the gap (G)2/mitotic (M) phase checkpoint during proximal tubule injury activates fibrotic cellular signals, such as transforming growth factor-β and connective tissue growth factor, which may contribute to the increased risk of CKD3,15–18. Epidemiologic data show that AKI may increase the risk of subsequent cardiovascular events by 1.5- to 2-fold3. While the definite mechanism is not clear, there is evidence that ischemia-reperfusion increases apoptosis in the heart, macrophage infiltrate, and angiotensin-converting enzyme expression19–21. There is also evidence that ischemia-reperfusion injury alters the affinity of the calcium receptor in the heart for inotropic agents22.\n\n\nPotential uses of AKI biomarkers\n\nNovel biomarkers of AKI are recent developments and typically reflect a specific component of AKI pathophysiology, including tubular injury, cell cycle arrest, systemic inflammatory pathways, and glomerular filtration (Figure 1). Biomarkers of AKI will hopefully have several uses in clinical care, including early diagnosis of AKI, prediction of clinical outcomes, and prediction of response to therapy. Initial investigations of biomarkers of AKI sought to determine if they could diagnose AKI earlier than serum creatinine, as it often takes 2 to 3 days before serum creatinine is elevated after a renal insult. Early diagnosis would be beneficial, as it can identify a window where clinicians could potentially intervene, such as by withdrawing nephrotoxic agents or providing treatment while injury is ongoing. If effective treatments for AKI are developed, clinicians could also potentially serially follow biomarkers of injury to determine if patients are responding to treatment. Evidence has also accumulated that biomarkers of AKI are associated with various patient outcomes, which is the focus of this review.\n\nAdapted from 73. AKI, acute kidney injury; H-FABP, heart fatty acid binding protein; IGFBP-7, insulin-like growth factor binding protein 7; IL-6, interleukin-6; IL-10, interleukin-10; IL-18, interleukin-18; KIM-1, kidney injury molecule-1; L-FABP, liver fatty acid binding protein; NAG, N-acetyl-β-D-glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; TIMP-2, tissue inhibitor metalloproteinase-2.\n\n\nBiomarkers of AKI and prognosis\n\nMany of the novel biomarkers of AKI are intricately involved in its pathogenesis, so it logically follows that they could be associated with adverse outcomes after AKI23–26. Biomarkers of AKI have been related to short- and long-term adverse outcomes in various patient care settings. The most commonly studied short-term outcomes are in-hospital mortality, need for renal replacement therapy (RRT), and length of stay. While there is evidence that biomarkers of AKI are related to long-term mortality, data are lacking to suggest that biomarkers of AKI are related to other important long-term patient outcomes, such as cardiovascular events and CKD, although prospective studies are ongoing27. Since AKI independently increases the risk of subsequent cardiovascular events and CKD, it follows that diagnostic biomarkers of AKI will also be associated with these poor outcomes. However, some AKI biomarkers may yield additional prognostic information beyond that provided by the AKI event itself. Long-term follow-up studies have identified a subgroup of patients who suffer from “subclinical AKI”. While these patients do not have AKI as defined by serum creatinine, they have elevated biomarkers of tubular injury and fare worse than patients without elevated biomarkers of AKI28,29. This suggests that biomarkers of AKI may provide additional prognostic information beyond that offered by serum creatinine. This observation has generated a new proposed framework for organizing AKI: structural versus functional AKI (Figure 2). This framework highlights two groups: the aforementioned patients with subclinical AKI and those with AKI as defined by serum creatinine but without any structural damage as indicated by low tubular injury markers, or “hemodynamic” AKI. This could prove meaningful, as it suggests that clinical trials investigating treatments for conditions such as acute tubular necrosis are enrolling patients unlikely to experience benefit (those with hemodynamic AKI) and failing to enroll patients who may benefit (those with subclinical AKI).\n\nAdapted from 74. AKI, acute kidney injury; GFR, glomerular filtration rate.\n\n\nUtility of novel biomarkers in various patient settings\n\nWhile novel biomarkers of AKI reflect the same pathophysiology regardless of the patient care setting, challenges for interpreting biomarkers of AKI differ between patient care settings, including cardiac surgery, critically ill, cardiorenal syndrome, transplant, and hospitalized patients.\n\nCardiac surgery is one of the most popular settings for the evaluation of biomarkers of AKI, partially because there is a clear point of injury. AKI after cardiac surgery has been commonly ascribed to an inflammatory cascade, although thus far interventions to quell the inflammatory cascade have been unsuccessful10. Biomarkers of AKI could help identify patients at higher risk of adverse outcomes.\n\nShort-term outcomes. One of the most carefully studied cohorts for biomarkers, Translational Research Investigating Biomarker Endpoints of AKI (TRIBE-AKI), featured over 1200 patients who underwent cardiac surgery. Levels of post-operative tubular injury biomarkers, such as urinary interleukin (IL)-18, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and liver fatty acid binding protein (L-FABP), were associated with increased risk of in-hospital death, increased length of stay, and need for RRT (Table 1)30,31. Patients with elevated urinary cystatin C, another tubular injury biomarker, additionally had increased risk of requiring RRT32.\n\n*p-value for trend significant, §p-value for trend not significant.\n\nAll values in ( ) are 95% confidence intervals, unless otherwise noted.\n\nAKI, acute kidney injury; AUC, area under the curve; HR, hazard ratio (3rd tertile compared to 1st tertile); IGFBP7, insulin like growth factor binding protein 7; IL-6, interleukin-6; IL-10, interleukin-10; IL-18, interleukin-18; IQR, interquartile range; KIM-1, kidney injury molecule-1; L-FABP, liver fatty acid binding protein; LOS, length of stay; NGAL, neutrophil gelatinase-associated lipocalin; Q1, quintile 1; Q5, quintile 5; RRT, renal replacement therapy; SD, standard deviation; TIMP-2, tissue inhibitor of metalloproteinases-2; d, days.\n\nAnother cohort of cardiac surgery patients, Southern Acute Kidney Injury Network (SAKInet), were evaluated to determine whether combinations of biomarkers could predict if patients with stage I AKI (AKIN) after cardiac surgery progressed to stage III AKI or death. Urinary IL-18 combined with percentage change in serum creatinine or urinary KIM-1 had the best discriminative ability to identify patients at high risk for progressing to higher stages of AKI or death within 30 days33. The SAKInet cohort study additionally evaluated urinary angiotensinogen and renin, biomarkers of intra-renal renin-angiotensin system activity, and found that they were associated with severe AKI or death within 30 days34.\n\nChanges in urinary biomarkers have been associated with improved patient outcomes. One study examined biomarkers that induce cell cycle arrest in cardiac surgery by examining the product of tissue inhibitor metalloproteinase 2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP-7). Differences in the decline of these biomarkers within 24 hours after surgery discriminated between patients who recovered and those who failed to recover from AKI35.\n\nLong-term outcomes. In the TRIBE-AKI study, patients with AKI as defined by serum creatinine and elevated peak post-operative tubular injury markers, including urinary IL-18, NGAL, KIM-1, and L-FABP, had an increased risk of long-term mortality28. Patients without AKI as defined by serum creatinine, elevated urinary IL-18, and KIM-1 also had an increase in long-term mortality of about 1.5-fold. These patients may have experienced subclinical AKI that was not detected by serum creatinine, leading to increased mortality, or it may be that urinary IL-18 or KIM-1 is capturing a systematic inflammatory process which increases the risk of death independently of AKI.\n\nBiomarkers that probe inflammatory pathways involved in AKI have additionally been investigated in TRIBE-AKI. Elevated first post-operative serum IL-10, which is known to attenuate the inflammatory cascade, was found to decrease the risk of long-term mortality36. Interestingly, this effect was isolated to patients with elevated urinary markers of tubular injury, which suggests that IL-10 protected those patients who were experiencing proximal tubular cell damage.\n\nAKI is a common complication in critical illness and is consistently associated with high mortality, although this is a challenging setting in which to study biomarkers of AKI1. There may be multiple renal insults, and the timing of renal insults is not always clear. Regardless, biomarker data have identified subphenotypes for complex intensive care unit (ICU) syndromes, such as acute respiratory distress syndrome (ARDS), which benefit from different treatment strategies37. The incorporation of prognostic information from AKI biomarkers could eventually lead to similar benefit. Multiple urinary biomarkers have been studied in critically ill patients and are associated with adverse outcomes, although the data are strongest for short-term outcomes (Table 2).\n\nAll values in ( ) are 95% confidence intervals, unless otherwise noted.\n\n*Outcomes at 9 months in patients with AKI § greater than cut-off versus less than cut-off † for 10-fold increase.\n\nAKI, acute kidney injury; AUC, area under the curve; HGF, hepatocyte growth factor; HR, hazard ratio (continuous unless otherwise noted); IL-18, interleukin-18; IGFBP7, insulin like growth factor binding protein 7; L-FABP, liver fatty acid binding protein; NAG, N-acetyl-beta-D-glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; OR, odds ratio; TIMP-2, tissue inhibitor of metalloproteinases-2.\n\nShort-term outcomes. One ICU cohort study, Validation of Biomarkers for Acute Lung Injury Diagnosis (VALID), comprising critically ill patients in both medical and surgical ICU settings, collected urinary biomarker specimens on admission and 48 hours after admission in over 400 patients. In VALID, admission levels of urinary IL-18 were independently associated with the composite outcome of 28-day mortality or dialysis even after adjustment for APACHE-II score, serum creatinine, and sepsis criteria38. Despite the relationship between urinary IL-18 and the composite outcome, other tubular injury markers, such as urinary NGAL and L-FABP, were associated only with the initiation of dialysis at 28 days and were not associated with the composite outcome39,40. This persisted despite the authors excluding patients with unidentified CKD or AKI upon enrollment into the study39. Similar results for urinary IL-18 and NGAL have been found in other cohorts of critically ill patients. A post hoc case-control study of 132 patients in the ARDS Network additionally found that increasing levels of urinary IL-18 on admission were associated with increased 1-month mortality41. Another prospective cohort of 339 patients in a mixed ICU setting showed that admission urinary L-FABP, IL-18, and NGAL could discriminate between survivors and non-survivors, and a combination of urinary NGAL and L-FABP was best able to discriminate between survivors and non-survivors42. Additionally, increasing levels of urinary cystatin C, a biomarker of glomerular filtration, in a group of 444 ICU patients were associated with increased risk of 1-month mortality43.\n\nAs in cardiac surgery, a decline in tubular injury biomarkers is also associated with improved patient outcomes. A post hoc analysis of 76 patients from the Acute Renal Failure Trial Network (ATN) trial, which evaluated the effect of dialysis dose on critically ill patients, investigated whether urinary biomarkers were associated with recovery from AKI and found that decreasing levels of urinary NGAL and hepatocyte growth factor (HGF) over a 2-week period predicted an increased chance of recovery of renal function 60 days after ICU admission44.\n\nLong-term outcomes. While biomarkers of tubular injury are studied most carefully, another cohort comprising 744 ICU patients (Sapphire) found that urinary biomarkers of cell cycle arrest (TIMP-2*IGFBP-7) were associated with a twofold increased risk of RRT and 9-month mortality, although this relationship was found only in patients with AKI as defined by serum creatinine45.\n\nHeart failure patients commonly suffer from AKI, which is strongly associated with increased mortality46. Cardiorenal syndrome, or the co-existence of AKI and heart failure, is often ascribed to hemodynamic perturbations or venous congestion. This is likely an over-simplification, as patients with cardiorenal syndrome respond differently to standard heart failure therapies, such as diuresis and inotropic support. Biomarker research has attempted to untangle the numerous phenotypes that exist within cardiorenal syndrome47.\n\nLong-term outcomes. There is extensive literature surrounding long-term prognosis and biomarkers of tubular injury in heart failure patients. Tubular injury biomarkers, including urinary KIM-1, NGAL, and N-acetyl-β-D-glucosaminidase (NAG), were associated with long-term mortality and recurrent heart failure hospitalization even after accounting for glomerular filtration rate (GFR) in multiple case-control studies and cohorts (Table 3)48–50. KIM-1 and NAG were significantly correlated with New York Heart Association class, suggesting that tubular injury may contribute to the pathophysiology of cardiorenal syndrome50. While urinary biomarkers of tubular injury have been consistently associated with mortality and other adverse outcomes, plasma NGAL has not demonstrated a consistent relationship, particularly after adjustment for renal function51–53. This has also been found in cardiac surgery and is likely because plasma NGAL is renally excreted, effectively rendering it another marker of GFR54.\n\nAll values in ( ) are 95% confidence intervals, unless otherwise noted.\n\n*unadjusted and 30 day follow-up, § greater than cut-off versus less than cut-off, † per 5U/gram Creatinine increase.\n\nAKI, acute kidney injury; HR, hazard ratio (per standard deviation increase unless otherwise noted); KIM-1, kidney injury molecule-1; MACE, major adverse cardiac events; NAG, N-acetyl-beta-D-glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin.\n\nNovel urinary biomarkers have also been investigated for prognostic utility in the kidney transplant setting and can have unique applications in the transplant field. Given the persistent shortage of organs and the increasing number of patients on the transplant list, biomarkers of AKI can potentially be used to decide if a deceased donor organ is acceptable for transplant as well as improve prediction for adverse recipient outcomes, such as delayed graft function (DGF) and graft failure (Table 4).\n\nAll values in ( ) are 95% confidence intervals, unless otherwise noted.\n\n*greater than cut-off versus less than cut-off, † per 100 ng/mg increase.\n\nAKI, acute kidney injury; H-FABP, heart fatty acid binding protein; HR, hazard ratio (continuous unless otherwise noted); IL-18, interleukin-18; KIM-1, kidney injury molecule-1; NAG, N-acetyl-beta-D-glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; OR, odds ratio.\n\nShort-term outcomes. In the perioperative period, the presence of urinary IL-18 and NGAL immediately after transplant was associated with an increased risk of DGF55,56. While the relationship between recipient kidney injury biomarkers and outcomes is relatively clear, the relationship between donor kidney injury biomarkers and recipient outcomes is more complex. One large multi-center cohort found that elevated donor urinary NGAL had a modest association with DGF in the recipient, although other tubular injury biomarkers were not significantly associated after multivariable adjustment57.\n\nLong-term outcomes. Single-center studies of transplant recipients found that elevated urinary tubular injury biomarkers, such as urinary IL-18, NGAL, NAG, and KIM-1, are associated with an increased risk of graft failure or death (Table 4)58,59. However, urinary donor biomarkers, including urinary IL-18, NGAL, and L-FABP, modified the relationship between DGF and 6-month recipient GFR. For recipients with DGF, higher levels of tubular injury biomarkers were associated with higher 6-month GFR, while for recipients who did not experience DGF, higher urinary donor biomarkers were associated with lower 6-month GFR57.\n\nWhile the most extensive evidence for the association between biomarkers of AKI and prognosis is in specific patient populations, biomarkers of AKI also have prognostic utility for short-term outcomes in heterogeneous populations. Multiple single-center cohorts of patients requiring nephrology consultation found that biomarkers of tubular injury were associated with an increased risk of in-hospital RRT or death (Table 5)60–63. Alternatively, elevated plasma NGAL, a marker of glomerular filtration, in patients hospitalized with pneumonia and severe AKI meant they were twice as likely to fail to recover renal function by discharge in adjusted analysis64. In another cohort of hospitalized patients requiring nephrology consultation owing to presumed acute tubular necrosis, elevated urinary epidermal growth factor levels allowed the authors to distinguish between patients who recovered renal function within 1 week of renal consult and those who did not. Additionally, elevated inflammatory cytokines identified patients who failed to recover renal function within 1 week of renal consult65. Urinary NGAL and KIM-1 measured at the time of emergency room visit were both independently associated with in-hospital RRT initiation or death after accounting for clinical parameters66.\n\nAll values in ( ) are 95% confidence intervals, unless otherwise noted.\n\n*4th quartile versus 1st–3rd quartile, § greater than cut-off versus less than cut-off, † per 300 ng/ml increase.\n\nAKI, acute kidney injury; AUC, area under the curve; EGF, epidermal growth factor; HR, hazard ratio; IL-18, interleukin-18; KIM-1, kidney injury molecule-1; NAG, N-acetyl-beta-D-glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; OR, odds ratio; RRT, renal replacement therapy.\n\n\nFuture directions\n\nThe bioinformatics revolution has improved discovery techniques for the identification of next-generation biomarkers. Biomarker research has primarily focused on biomarkers that are considered part of the proteome. Recent studies have begun examining other layers of cellular data, including microRNA and mitochondrial DNA67–70. While these biomarkers are in the early stages of investigation, they hold great promise, as they provide opportunities to probe other components of cellular pathways.\n\nSeveral barriers remain for the clinical application of biomarkers despite the extensive evidence that they are associated with prognosis. Biomarkers of AKI often provide modest improvement in discriminative ability compared to traditional clinical models and have different trajectories and time scales depending upon the patient population and biomarker being studied. This will pose a challenge for busy clinicians attempting to incorporate a cornucopia of biomarkers into clinical practice. Moreover, data are lacking to support whether biomarkers are associated with organ-specific long-term outcomes, such as CKD or cardiovascular disease. Technical challenges also exist, as developing, standardizing, and validating biomarker assays is a laborious process71. Most importantly, researchers have yet to identify therapeutic changes to institute once patients are identified as being at high risk for adverse outcomes based on biomarkers of AKI.\n\nGiven the heterogeneity and dynamic nature of AKI, one biomarker will not accurately predict all adverse outcomes across several settings, nor will it accurately identify the various stages in the natural history of AKI. One important task is to definitively identify the natural stages of AKI as demarcated by biomarkers: injury, maintenance, and repair. This could help create a more balanced picture of prognostic outcomes for patients. For example, patients may have different outcomes depending on the magnitude of “injury” biomarkers balanced by the level of “repair” biomarkers.\n\nWhile challenges remain, biomarkers can offer several potential benefits to the diagnosis, management, and treatment of AKI in the foreseeable future. Perhaps the most immediate application is to identify patients who are experiencing renal injury from nephrotoxic agents, prolonged surgeries, cardiorenal syndrome, or hepatorenal syndrome. Patients with elevated injury or fibrosis markers could be considered for alternative therapy instead of waiting for these patients to develop AKI or CKD as defined by serum creatinine. However, for biomarkers to become the “standard of care” for the management of these patients, prospective trials should be conducted to identify cut-points for biomarker levels which will identify patients at a sufficiently high risk of renal complications to warrant providing a therapeutic intervention or adjusting surgical management.\n\nBiomarkers of AKI can have two potential applications in clinical trials of putative drug treatments for AKI. First, they will allow us to identify patients who have ongoing injury so that therapies may be provided while the injury process is occurring. Second, the greatest potential for biomarkers is if they can identify patients at high risk of adverse outcomes. In such a scenario, treatment can then be personalized for high-risk subgroups of patients. Trials in AKI have been hindered by low event rates and are likely enrolling patients with multiple phenotypes of AKI. Given the wealth of information showing that biomarkers are related to important patient outcomes, such as RRT and death, future clinical trials for AKI should enroll patients with elevated biomarkers of AKI in order to increase the likelihood that patients would actually benefit from the putative treatment. The TOPCAT trial, which examined the benefit of spironolactone in heart failure patients with preserved ejection fraction, found that treatment benefit was isolated to patients with elevated natriuretic peptide levels72. Future AKI trials should employ similar enrollment strategies, as treatment benefit may be isolated to particular AKI phenotypes. The Assessment, Serial Evaluation and Subsequent Sequelae of Acute Kidney Injury study is ongoing to determine if patients with AKI experience increased risk of long-term complications, such as cardiovascular events and CKD27. This study should help ascertain if it is worthwhile enrolling patients with AKI as defined by biomarkers in lieu of serum creatinine in future clinical trials.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nChirag R. Parikh is supported by the NIH grants K24DK090203, P30 DK079310 O'Brien Center Grant and R01HL085757. Jennifer A. Schaub is supported by T32DK007276-36.\n\n\nReferences\n\nUchino S, Kellum JA, Bellomo R, et al.: Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA. 2005; 294(7): 813–8. PubMed Abstract | Publisher Full Text\n\nBagshaw SM, Uchino S, Bellomo R, et al.: Septic acute kidney injury in critically ill patients: clinical characteristics and outcomes. Clin J Am Soc Nephrol. 2007; 2(3): 431–9. PubMed Abstract | Publisher Full Text\n\nCoca SG, Yusuf B, Shlipak MG, et al.: Long-term risk of mortality and other adverse outcomes after acute kidney injury: a systematic review and meta-analysis. Am J Kidney Dis. 2009; 53(6): 961–73. 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PubMed Abstract | Publisher Full Text\n\nGo AS, Parikh CR, Ikizler TA, et al.: The assessment, serial evaluation, and subsequent sequelae of acute kidney injury (ASSESS-AKI) study: design and methods. BMC Nephrol. 2010; 11: 22. PubMed Abstract | Free Full Text\n\nCoca SG, Garg AX, Thiessen-Philbrook H, et al.: Urinary biomarkers of AKI and mortality 3 years after cardiac surgery. J Am Soc Nephrol. 2014; 25(5): 1063–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaase M, Devarajan P, Haase-Fielitz A, et al.: The outcome of neutrophil gelatinase-associated lipocalin-positive subclinical acute kidney injury: a multicenter pooled analysis of prospective studies. J Am Coll Cardiol. 2011; 57(17): 1752–61. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nParikh CR, Coca SG, Thiessen-Philbrook H, et al.: Postoperative biomarkers predict acute kidney injury and poor outcomes after adult cardiac surgery. J Am Soc Nephrol. 2011; 22(9): 1748–57. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nParikh CR, Thiessen-Philbrook H, Garg AX, et al.: Performance of kidney injury molecule-1 and liver fatty acid-binding protein and combined biomarkers of AKI after cardiac surgery. Clin J Am Soc Nephrol. 2013; 8(7): 1079–88. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoyner JL, Garg AX, Shlipak MG, et al.: Urinary cystatin C and acute kidney injury after cardiac surgery. Am J Kidney Dis. 2013; 61(5): 730–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArthur JM, Hill EG, Alge JL, et al.: Evaluation of 32 urine biomarkers to predict the progression of acute kidney injury after cardiac surgery. Kidney Int. 2014; 85(2): 431–8. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nAlge JL, Karakala N, Neely BA, et al.: Association of elevated urinary concentration of renin-angiotensin system components and severe AKI. Clin J Am Soc Nephrol. 2013; 8(12): 2043–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeersch M, Schmidt C, van Aken H, et al.: Urinary TIMP-2 and IGFBP7 as early biomarkers of acute kidney injury and renal recovery following cardiac surgery. PLoS One. 2014; 9(3): e93460. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang WR, Garg AX, Coca SG, et al.: Plasma IL-6 and IL-10 Concentrations Predict AKI and Long-Term Mortality in Adults after Cardiac Surgery. J Am Soc Nephrol. 2015; 26(12): 3123–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCalfee CS, Delucchi K, Parsons PE, et al.: Subphenotypes in acute respiratory distress syndrome: latent class analysis of data from two randomised controlled trials. Lancet Respir Med. 2014; 2(8): 611–20. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSiew ED, Ikizler TA, Gebretsadik T, et al.: Elevated urinary IL-18 levels at the time of ICU admission predict adverse clinical outcomes. Clin J Am Soc Nephrol. 2010; 5(8): 1497–505. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSiew ED, Ware LB, Bian A, et al.: Distinct injury markers for the early detection and prognosis of incident acute kidney injury in critically ill adults with preserved kidney function. Kidney Int. 2013; 84(4): 786–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSiew ED, Ware LB, Gebretsadik T, et al.: Urine neutrophil gelatinase-associated lipocalin moderately predicts acute kidney injury in critically ill adults. J Am Soc Nephrol. 2009; 20(8): 1823–32. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nParikh CR, Abraham E, Ancukiewicz M, et al.: Urine IL-18 is an early diagnostic marker for acute kidney injury and predicts mortality in the intensive care unit. J Am Soc Nephrol. 2005; 16(10): 3046–52. PubMed Abstract | Publisher Full Text\n\nDoi K, Negishi K, Ishizu T, et al.: Evaluation of new acute kidney injury biomarkers in a mixed intensive care unit. Crit Care Med. 2011; 39(11): 2464–9. PubMed Abstract | Publisher Full Text\n\nNejat M, Pickering JW, Walker RJ, et al.: Urinary cystatin C is diagnostic of acute kidney injury and sepsis, and predicts mortality in the intensive care unit. Crit Care. 2010; 14(3): R85. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSrisawat N, Wen X, Lee M, et al.: Urinary biomarkers and renal recovery in critically ill patients with renal support. Clin J Am Soc Nephrol. 2011; 6(8): 1815–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoyner JL, Shaw AD, Chawla LS, et al.: Tissue Inhibitor Metalloproteinase-2 (TIMP-2)·IGF-Binding Protein-7 (IGFBP7) Levels Are Associated with Adverse Long-Term Outcomes in Patients with AKI. J Am Soc Nephrol. 2015; 26(7): 1747–54. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSmith GL, Lichtman JH, Bracken MB, et al.: Renal impairment and outcomes in heart failure: systematic review and meta-analysis. J Am Coll Cardiol. 2006; 47(10): 1987–96. PubMed Abstract | Publisher Full Text\n\nBrisco MA, Testani JM: Novel renal biomarkers to assess cardiorenal syndrome. Curr Heart Fail Rep. 2014; 11(4): 485–99. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDamman K, Masson S, Hillege HL, et al.: Clinical outcome of renal tubular damage in chronic heart failure. Eur Heart J. 2011; 32(21): 2705–12. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDamman K, van Veldhuisen DJ, Navis G, et al.: Tubular damage in chronic systolic heart failure is associated with reduced survival independent of glomerular filtration rate. Heart. 2010; 96(16): 1297–302. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJungbauer CG, Birner C, Jung B, et al.: Kidney injury molecule-1 and N-acetyl-β-D-glucosaminidase in chronic heart failure: possible biomarkers of cardiorenal syndrome. Eur J Heart Fail. 2011; 13(10): 1104–10. PubMed Abstract | Publisher Full Text\n\nMaisel AS, Mueller C, Fitzgerald R, et al.: Prognostic utility of plasma neutrophil gelatinase-associated lipocalin in patients with acute heart failure: the NGAL EvaLuation Along with B-type NaTriuretic Peptide in acutely decompensated heart failure (GALLANT) trial. Eur J Heart Fail. 2011; 13(8): 846–51. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nShrestha K, Borowski AG, Troughton RW, et al.: Renal dysfunction is a stronger determinant of systemic neutrophil gelatinase-associated lipocalin levels than myocardial dysfunction in systolic heart failure. J Card Fail. 2011; 17(6): 472–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Deursen VM, Damman K, Voors AA, et al.: Prognostic value of plasma neutrophil gelatinase-associated lipocalin for mortality in patients with heart failure. Circ Heart Fail. 2014; 7(1): 35–42. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMoledina DG, Parikh CR, Garg AX, et al.: Association of Perioperative Plasma Neutrophil Gelatinase-Associated Lipocalin Levels with 3-Year Mortality after Cardiac Surgery: A Prospective Observational Cohort Study. PLoS One. 2015; 10(6): e0129619. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHall IE, Yarlagadda SG, Coca SG, et al.: IL-18 and urinary NGAL predict dialysis and graft recovery after kidney transplantation. J Am Soc Nephrol. 2010; 21(1): 189–97. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nParikh CR, Jani A, Mishra J, et al.: Urine NGAL and IL-18 are predictive biomarkers for delayed graft function following kidney transplantation. Am J Transplant. 2006; 6(7): 1639–45. PubMed Abstract | Publisher Full Text\n\nReese PP, Hall IE, Weng FL, et al.: Associations between Deceased-Donor Urine Injury Biomarkers and Kidney Transplant Outcomes. J Am Soc Nephrol. 2016; 27(5): 1534–43. PubMed Abstract | Publisher Full Text\n\nHall IE, Doshi MD, Reese PP, et al.: Association between peritransplant kidney injury biomarkers and 1-year allograft outcomes. Clin J Am Soc Nephrol. 2012; 7(8): 1224–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNauta FL, Bakker SJ, van Oeveren W, et al.: Albuminuria, proteinuria, and novel urine biomarkers as predictors of long-term allograft outcomes in kidney transplant recipients. Am J Kidney Dis. 2011; 57(5): 733–43. PubMed Abstract | Publisher Full Text\n\nHall IE, Coca SG, Perazella MA, et al.: Risk of poor outcomes with novel and traditional biomarkers at clinical AKI diagnosis. Clin J Am Soc Nephrol. 2011; 6(12): 2740–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiangos O, Perianayagam MC, Vaidya VS, et al.: Urinary N-acetyl-beta-(D)-glucosaminidase activity and kidney injury molecule-1 level are associated with adverse outcomes in acute renal failure. J Am Soc Nephrol. 2007; 18(3): 904–12. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nNejat M, Pickering JW, Devarajan P, et al.: Some biomarkers of acute kidney injury are increased in pre-renal acute injury. Kidney Int. 2012; 81(12): 1254–62. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSinger E, Elger A, Elitok S, et al.: Urinary neutrophil gelatinase-associated lipocalin distinguishes pre-renal from intrinsic renal failure and predicts outcomes. Kidney Int. 2011; 80(4): 405–14. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSrisawat N, Murugan R, Lee M, et al.: Plasma neutrophil gelatinase-associated lipocalin predicts recovery from acute kidney injury following community-acquired pneumonia. Kidney Int. 2011; 80(5): 545–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKwon O, Ahn K, Zhang B, et al.: Simultaneous monitoring of multiple urinary cytokines may predict renal and patient outcome in ischemic AKI. Ren Fail. 2010; 32(6): 699–708. PubMed Abstract | Publisher Full Text\n\nNickolas TL, Schmidt-Ott KM, Canetta P, et al.: Diagnostic and prognostic stratification in the emergency department using urinary biomarkers of nephron damage: a multicenter prospective cohort study. J Am Coll Cardiol. 2012; 59(3): 246–55. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLee CG, Kim JG, Kim HJ, et al.: Discovery of an integrative network of microRNAs and transcriptomics changes for acute kidney injury. Kidney Int. 2014; 86(5): 943–53. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRamachandran K, Saikumar J, Bijol V, et al.: Human miRNome profiling identifies microRNAs differentially present in the urine after kidney injury. Clin Chem. 2013; 59(12): 1742–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaikumar J, Hoffmann D, Kim TM, et al.: Expression, circulation, and excretion profile of microRNA-21, -155, and -18a following acute kidney injury. Toxicol Sci. 2012; 129(2): 256–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhitaker RM, Stallons LJ, Kneff JE, et al.: Urinary mitochondrial DNA is a biomarker of mitochondrial disruption and renal dysfunction in acute kidney injury. Kidney Int. 2015; 88(6): 1336–44. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPepe MS, Etzioni R, Feng Z, et al.: Phases of biomarker development for early detection of cancer. J Natl Cancer Inst. 2001; 93(14): 1054–61. PubMed Abstract | Publisher Full Text\n\nPitt B, Pfeffer MA, Assmann SF, et al.: Spironolactone for heart failure with preserved ejection fraction. N Engl J Med. 2014; 370(15): 1383–92. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKoyner JL, Parikh CR: Clinical utility of biomarkers of AKI in cardiac surgery and critical illness. Clin J Am Soc Nephrol. 2013; 8(6): 1034–42. PubMed Abstract | Publisher Full Text\n\nHuen SC, Parikh CR: Molecular phenotyping of clinical AKI with novel urinary biomarkers. Am J Physiol Renal Physiol. 2015; 309(5): F406–13. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "13963",
"date": "24 May 2016",
"name": "Charuhas V. Thakar",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13964",
"date": "24 May 2016",
"name": "Brian Reeves",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-986
|
https://f1000research.com/articles/5-40/v1
|
08 Jan 16
|
{
"type": "Opinion Article",
"title": "The dream of health information for all",
"authors": [
"Alvaro Proaño",
"Eloy F Ruiz",
"Ruben Porudominsky",
"Jose Carlos Tapia",
"Eloy F Ruiz",
"Ruben Porudominsky",
"Jose Carlos Tapia"
],
"abstract": "In 2004, an influential report in The Lancet suggested that open health information for all could be achieved by 2015. Unfortunately, this goal has not yet been accomplished. Despite progress in obtaining quality scientific articles in Latin America, it remains difficult to reliably access new and cutting-edge research. As graduating Peruvian medical students, we have confronted many obstacles in obtaining access to quality and up-to-date information and a constant tension between accessing \"what is available\" rather than \"what we need\". As we have learned, these limitations affect not only our own education but also the choices we make in the management of our patients. In the following article, we state our point of view regarding limitations in access to scientific articles in Peru and Latin America.",
"keywords": [
"Publishing",
"Education",
"Communication",
"Peru",
"Medical Students"
],
"content": "Introduction\n\nSince the earliest days of scientific publishing, the dissemination of research has been intended to maximize impact rather than profit1. Although this philosophy remains with the scientific community to this day, for-profit publishers continue to charge for access. However, with the development of the internet, a new hope was born for open access2.\n\nIn 2004, Godlee et al. proposed in The Lancet, that we could achieve health information for all by 20153; nevertheless, eleven years later this goal has not yet been fulfilled. Instead, the open medical repository is just a fraction of all the information out there. For example, during 2011, only 17% of the 1.66 million articles published and indexed in Scopus were available through open access4.\n\nFull access to scholarly articles carries several benefits to researchers, healthcare professionals, policymakers and to society in general5,6. The alternative scenario results in low literacy for medical professionals, affecting the diagnosis and treatment of patients. The scarcity of open literature contributes to an out-of-date medical practice, where health professionals “[believe] that they can go about their job without new information”7. This type of practice is unacceptable and should be changed for the well-being of our patients.\n\nMedical school is a key moment to learn how to read critically the current research and to keep up-to-date with it. If we learn it as students, we will carry this practice throughout our career, but if we do not, it will only become harder later. In this context, after having gone through seven years of medical school, we believe it is important to state our point of view, as medical students, regarding the limitation of access to scientific articles in Peru and Latin America.\n\n\nAccess to research publications in Latin America\n\nNowadays, without any doubt, the two most visible Latin American efforts to achieve the dream of open access and to catalyze globalized science are the Scientific Electronic Library Online (SciELO), and The RedALyC project (Red de Revistas Científicas de América Latina y el Caribe, España y Portugal)8–10. SciELO was conceived in 1997 as a pilot program to create a virtual library of Brazilian biomedical journals8,9. This later became an expanding publishing platform for Latin American journals that currently includes 15 countries from Latin America, Europe and Africa9. RedALyC, established in 2003, was born out as a need to cover the social sciences and humanities (not initially covered by SciELO) that also widened its original focus to all disciplines8. Free-full-text articles in this region have prevailed because most of the journals are not-for-profit enterprises maintained by public academic institutions or scientific societies. In contrast to the usual ‘article publishing charge’ model of open access, Latin American journals follow a ‘fee-less-free’ model providing immediate free access to the electronic edition of the journals without charging the author’s institution for submission, processing or publication of the article on behalf of medical societies or associations8,9,11. This enables greater visibility for thousands of articles from Latin America10.\n\nLatin America has long been active in promoting the visibility of its scientific production9. This has been reflected in several initiatives such as the Network of Virtual Libraries of the Latin American Council of Social Sciences (CLACSO), the Alliance of Agricultural Information Services (SIDALC), and LATINDEX, all created before the Budapest Open Access Initiative in 200210. Significantly, LATINDEX was one of the first referencing systems for the Ibero-American region, indexing journals related to social and medical sciences9. As Vessuri et al. stated, “[these are clearly great examples] of a region that was already exploring the possibility of building national collections of full-text electronic journals in open access”9. The most recent open access initiative in the region is the Federated Network of Institutional Repositories of Scientific Publications (LA Referencia)10. The aspiration of this regional network is to become the principal gateway through which Latin America expresses its scientific production to the rest of the world12. Since it was established in 2012, this interoperable network of national repository systems (including Argentina, Brazil, Chile, Colombia, Ecuador, El Salvador, México, Peru and Venezuela) has gathered more than 800,000 full-text documents available for all10,12.\n\nDespite the Latin American contribution towards policies in favor of open access, there is still scarce evidence to guarantee the fulfillment of “universal access to essential health-care information”3. In other words, even though information is free in SciELO and RedALyC, most of the journals indexed by these services are outdated. A recent analysis in the field of Dermatology found that 60% of journals in these databases were not recent, had lower impact factors and published fewer issues per year when compared to non-free journals indexed in MEDLINE/Pubmed11.\n\nOutside of the continent, other international initiatives have also recently been developed. Open science publishing platforms, such as the Public Library of Science (PLoS), BioMed Central (BMC), PeerJ or F1000Research2,13 allow scientists to publish their work in a high quality platform that also make their content freely available. Additionally, since 2005, research-funding agencies, such as the Wellcome Trust (UK) and National Institutes of Health (USA), promote a policy that all funded work should be publicly available and provide funds to cover the costs of publishing in open access journals14. These collaborative strategies are increasingly allowing healthcare professionals from developing countries to gain free access to high quality research.\n\nMoreover, there are other global alliances that have also aimed to diminish the gap between what exists and what is available. HINARI15, for example, is one of the four programs from Research4Life, a public-private partnership of the World Health Organization, whose main goal is to provide “free or low cost access to academic and professional peer-reviewed content online”16. HINARI has provided access to more than 13,000 journals to developing countries since it was launched in 2002 and has opened the door to a world of relevant and latest medical information5,16. However, many top-notch journals are not always accessible through HINARI, which in turn creates a major setback for medical students17.\n\n\nAccess to research publications in Peru\n\nIn Peru, the main sources of scientific information available for students and healthcare professionals are journals indexed in the Latin American databases mentioned above. The access to other international journals, such as those indexed in MEDLINE, is limited by the agreements of each institute. Still, there are two Peruvian journals indexed in MEDLINE and both are characterized by providing articles online free of cost. These journals are the Revista Peruana de Medicina Experimental y Salud Publica (http://bit.do/rpmesp), and the Revista de Gastroenterología del Perú (http://bit.do/revgastrope). All articles from these journals follow a ‘fee-less-free’ model and are deposited in their respective SciELO library, enabling flow of information and greater visibility18.\n\nIn addition, Peruvian universities have developed strategies to keep their scientific production visible, available online and free of cost. We will mention three examples of these initiatives. Some universities require students to finish their career with a thesis that is then stored online in the online CyberTesis repository. This project was created to allow all theses to stay available online; however, this project is run independently by each school. Universities also possess their own independent journals and offer online repositories for the publications found therein. University-run journals can also upload their publications to SciELO repositories, like the Revista Medica Herediana (http://bit.do/revmedhered) from the Universidad Peruana Cayetano Heredia. Finally, as in other parts of the world19, Open Access Week is celebrated locally by some Universities such as Pontificia Universidad Católica del Perú20 and Universidad de Lima21.\n\nThe government of Peru also promotes open access. Under the Peruvian Law N° 30035, there must be a national open-access repository managed by the National Council of Science and Technology and Technological Innovation (CONCYTEC)22. This repository provides access to scientific productions developed in our country (http://alicia.concytec.gob.pe/vufind/) and also grants access to scientific databases such as ScienceDirect, EBSCO and ProQuest through its virtual library (http://proyectos.concytec.gob.pe/access/). However, this initiative is not well known among students and entails several registration requirements that are difficult for students to fulfill.\n\n\nOur perspective\n\nWe have completed seven years of medical education in Peru, and as we have passed through each stage of basic science, pre-clinical and clinical work, our need for updated knowledge has increased. This process has a huge impact on our training because medical school is the period during which we are expected to learn to critically read and to keep up-to-date with current clinical research. Our ability to master this skill then reflects on our clinical decision making and ultimately, affects our patients. Therefore, access to information is not only an intellectual exercise, but also an ethical obligation of our profession. If we ever expect to improve the lives of our patients, we need “up-to-date, easy, fast, reliable, and affordable accesses to scientific information”23.\n\nIn 2007, Villafuerte-Gálvez et al. (as a Peruvian medical student) suggested that students in low-income countries have a difficult time maintaining and advancing their knowledge and skills due to the limited access to pay-per-view journals17. This could become a threat to the student’s education due to the fact that most of these journals require readers to pay a fee at an unattainable cost (USD20–USD45) for the majority of medical students in developing countries17, making access to research a “pay-wall” to overcome19.\n\nIt has been shown that at least one in three recent Peruvian medical graduates use MEDLINE/PubMed to search for scientific articles24; however, we found no data on how many of these queries achieve access to full-text articles. In our experience, “only through removing the barriers to access to global research that health improvements can be accelerated”25. The problem we hope to tackle some day is the one related to the “the ‘last mile’ of the process which actually delivers the document or other source that has been searched for”26.\n\nIn order to obtain the best medical knowledge, as medical students from a developing country, we must overcome two main obstacles: access and language. Even if barriers to access can be overcome, the language barrier may remain. English is considered the language of biomedical science. However, not all scientists know it, especially in rural and developing areas. It is a shame when researchers studying the health problems of a rural area only report their findings in English. How will the healthcare personnel from that area learn and apply this knowledge? Some Latin American journals have aimed to lessen this gap by providing the same article, with the same digital object identifier (DOI), in more than one language. This is obviously extra work for both the publisher and the authors, but if the information cannot be understood, then it will not be applied.\n\nAs The Finch Group Report suggests, strategies that aim to achieve the dream of “Health information for all” should be guided by four principles: access, usability, quality, and cost and sustainability27. First, research should be freely accessible to individuals and organizations all around the globe. As mentioned earlier, the key issue about this criterion is the availability of free full-text, ideally in the native language where the research was done. Second, usability implies being able to access the full range of the latest tools to organize, analyze and manipulate the content relevant to their work. Third, the rigorous peer-review process must be maintained without affecting the previous principles. The openness and transparency of academic publishers (not-for-profit) guarantee the quality and development of publications supported by peer-review. Finally, citing The Finch Report, “the main point of cost and sustainability is that no form of publishing is cost-free, and the key requirement is therefore that publishers, whether commercial or not-for-profit, should be able to generate revenues to meet the cost of services they provide”27.\n\nEleven years have passed and despite some initiatives, the goal of open access has not been achieved. We have not lost hope that universal access to essential health-care information will occur, but maybe is time to change our individual approach and for governments, universities and academic societies to develop a unified strategy by which open access can be achieved, while still fulfilling the four principles aforementioned.\n\nWill we keep dealing with the dilemma of accessing “what is available” rather than “what we need”?",
"appendix": "Author contributions\n\n\n\nAP, EFR, RP and JCT conceived the study and prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe would like to thank Dr. Gwenyth Lee and Gaby Caro for their comments and suggestions.\n\n\nReferences\n\nAlbert KM: Open access: implications for scholarly publishing and medical libraries. J Med Libr Assoc. 2006; 94(3): 253–262. PubMed Abstract | Free Full Text\n\nBjörk BC: Have the \"mega-journals\" reached the limits to growth? PeerJ. 2015; 3: e981. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGodlee F, Pakenham-Walsh N, Ncayiyana D, et al.: Can we achieve health information for all by 2015? Lancet. 2004; 364(9430): 295–300. PubMed Abstract | Publisher Full Text\n\nLaakso M, Björk BC: Anatomy of open access publishing: a study of longitudinal development and internal structure. BMC Med. 2012; 10: 124. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAronson B: Improving online access to medical information for low-income countries. N Engl J Med. 2004; 350(10): 966–8. PubMed Abstract | Publisher Full Text\n\nPakenham-Walsh N, Priestley C, Smith R: Meeting the information needs of health workers in developing countries. BMJ. 1997; 314(7074): 90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKale R: Health information for the developing world. BMJ. 1994; 309(6959): 939–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAguado-López E, Garduño-Oropeza GA, Rogel-Salazar R, et al.: The need and viability of a mediation index in Latin American scientific production and publication: The case of the Redalyc System of Scientific Information. Aslib Proc. 2012; 64(1): 8–31. Publisher Full Text\n\nVessuri H, Guédon J, Cetto AM: Excellence or quality? Impact of the current competition regime on science and scientific publishing in Latin America and its implications for development. Curr Sociol. 2014; 62(5): 647–665. Publisher Full Text\n\nBabini D, Machin-Mastromatteo JD: Latin American science is meant to be open access: Initiatives and current challenges. Inf Development. 2015; 31(5): 477–481. Publisher Full Text\n\nTapia JC, Diaz LA, Bravo F: Dermatological journals available to Latin American dermatologists. Int J Dermatol. 2015; 54(11): 1333–7. PubMed Abstract | Publisher Full Text\n\nRed Federada de Repositorios Institucionales de Publicaciones Científicas (LA Referencia): América Latina pasa la primera página en Acceso Abierto. Red Clara & Banco Interamericano de Desarrollo, [Last accessed on 2015 Dec 12]. Reference Source\n\nWhite E: New Journals that are changing the way we publish. [Last accessed on 2015 Dec 12]. Reference Source\n\nVan Noorden R: Funders punish open-access dodgers. Nature. 2014; 508(7495): 161. PubMed Abstract | Publisher Full Text\n\nResearch4Life. [Last accessed on 2015 Dec 12]. Reference Source\n\nHINARI. [Last accessed on 2015 Dec 12]. Reference Source\n\nVillafuerte-Gálvez J, Curioso WH, Gayoso O: Biomedical journals and global poverty: is HINARI a step backwards? PLoS Med. 2007; 4(6): e220. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuamaní C, Pacheco-Romero J: [Visibility and productivity of the biomedical Peruvian journals]. Rev Gastroenterol Peru. 2009; 29(2): 132–9. PubMed Abstract\n\nMatheka DM, Nderitu J, Mutonga D, et al.: Open access: academic publishing and its implications for knowledge equity in Kenya. Global Health. 2014; 10: 26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOpen Access Perú: Acceso Abierto a la Información Académica y Científica en el Perú. Pontificia Universidad Católica del Perú. [Last accessed on 2015 Dec 12]. Reference Source\n\nUniversidad de Lima: Semana Internacional del Open Access. 2014. [Last accessed on 2015 Dec 12]. Reference Source\n\nCONCYTEC: Ley que regula el repositorio nacional digital de ciencia, tecnología e innovación de acceso abierto. [Last accessed on 2015 Dec 12]. Reference Source\n\nLindberg DA, Humphreys BL: Rising expectations: access to biomedical information. Yearb Med Inform. 2008; 3(1): 165–72. PubMed Abstract | Free Full Text\n\nMejia CR, Caceres OJ, Vera CA, et al.: [Use of information sources by recently graduated physicians of Lima]. Rev Peru Med Exp Salud Publica. 2014; 31(4): 716–20. PubMed Abstract\n\nChan L, Arunachalam S, Kirsop B: Open access: a giant leap towards bridging health inequities. Bull World Health Organ. 2009; 87(8): 631–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nResearch Information Network. Overcoming barriers: access to research information content. UK, 2009; [Last accessed on 2015 Dec 12]. Reference Source\n\nFinch DJ: Accessibility, sustainability, excellence: how to expand access to research publications. Report of the Working Group on Expanding Access to Published Research Findings, 2012; [Last accessed on 2015 Dec 12]. Reference Source"
}
|
[
{
"id": "12114",
"date": "01 Feb 2016",
"name": "Lorna Wildgaard",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nProaño et al. present an interesting opinion paper about the availability of health information for health practitioners in Latin America. The title gains in irony after one reads the article, which concludes that health information for all remains a dream and not an accomplishable goal. The abstract clearly contextualizes the article to the opinions of the authors and adequately encapsulates the depth of the subject matter. The arguments presented in the article are clearly stated as opinions based on personal experiences, and thus are not necessarily substantiated by evidence gathered from relevant literature. I recommend the article be approved as an opinion paper – this is not ground breaking new knowledge - but is a topic that needs discussion as in the end, access to information can affect clinical decisions.I recommend the following minor revisions:p.3, column 1, line 18-20. “This project was created to allow all theses to stay available online: however, this project is run independently by each school.”Please include a sentence on why this is problematic – is it a meta-data problem, incompleteness, etc. Do the authors have a solution?p.3, column 2, line 1. “In 2007, Villafuerte-Gálvez et al. (as a Peruvian medical student)Please correct the use of the singular to the plural eg (as Peruvian medical students)p3. Column 2, line 9-12. “It has been shown that at least one in three recent Peruvian medical graduates use Medline/PubMed….”Is this surprisingly different from any other country? In the referenced article by Mejia et al. (ref.#24) 34% of the sample regularly used Medline/PubMed. Other studies, using other countries, for example by Davis (2011, follow the link below), report use of PubMed/MEDLINE to be between 70 and 84%.https://www.researchgate.net/profile/Karen_Davies/publication/49763179_Physicians_and_their_use_of_information_A_survey_comparison_between_the_United_States_Canada_and_the_United_Kingdom/links/004635286b7abf103d000000.pdf p.3, column 2, line 51. “…but maybe is time to change..”Please correct to “…but maybe it is time to change…”p4, column 1, line 2. “ …fulfilling the four principles aforementioned.”Please change to “…fulfilling the aforementioned four principles.”p.4, column 1, line 3-4. “Will we keep dealing with the dilemma of accessing “what is available” rather than “what we need”?The ending could be stronger. Please consider rewriting this final sentence. Try something like: “Until then, we will keep dealing with the dilemma of accessing “what is available” rather than “what we need”.For my personal interest:p.3, column 2, line 26-28 “Some Latin American journals have aimed to lessen the gap by providing the same article, with the same digital object identifier, in more than one language”This is interesting, I would like to learn more. Is this allowed, ethically? Is this not self-plagiarism? How do you cite such publications and do they count as one or two separate works?",
"responses": [
{
"c_id": "1903",
"date": "24 May 2016",
"name": "Alvaro Proaño",
"role": "Author Response",
"response": "Thank you very much for your suggestions and for taking the time to review our paper. We look forward to any comments you have on this in the future. Response to specific comments is as follows:p.3, column 1, line 18-20. “This project was created to allow all theses to stay available online: however, this project is run independently by each school.”Please include a sentence on why this is problematic – is it a meta-data problem, incompleteness, etc. Do the authors have a solution?Response: “This is a problem because its not a centralized archive but instead independent local ones. Researchers like to look for information in centralized databases, or at least use a meta-searcher. Local universities should join initiatives to centralize and share their repositories making it easier for researchers to read and cite them.”p.3, column 2, line 1. “In 2007, Villafuerte-Gálvez et al. (as a Peruvian medical student)Please correct the use of the singular to the plural eg (as Peruvian medical students)Response: Change madep3. Column 2, line 9-12. “It has been shown that at least one in three recent Peruvian medical graduates use Medline/PubMed….”Is this surprisingly different from any other country? In the referenced article by Mejia et al. (ref.#24) 34% of the sample regularly used Medline/PubMed. Other studies, using other countries, for example by Davis (2011, follow the link below), report use of PubMed/MEDLINE to be between 70 and 84%.https://www.researchgate.net/profile/Karen_Davies/publication/49763179_Physicians_and_their_use_of_information_A_survey_comparison_between_the_United_States_Canada_and_the_United_Kingdom/links/004635286b7abf103d000000.pdfResponse: We agree with your point of view and think that contrasting Peruvian reality with the one in the UK brings about a good comparison for readers to understand Peru’s reality. Thus, it now reads: “It has been shown that at least one in three (33%) recent Peruvian medical graduates use MEDLINE/PubMed to search for scientific articles 24 which is much lower than that reported in physicians from USA (81%), Canada (76%) or UK (77%) add new citation; however, we found no data on how many of these queries achieve access to full-text articles.”p.3, column 2, line 51. “…but maybe is time to change..”Please correct to “…but maybe it is time to change…”Response: Change madep4, column 1, line 2. “ …fulfilling the four principles aforementioned.”Please change to “…fulfilling the aforementioned four principles.”Response: Change madep.4, column 1, line 3-4. “Will we keep dealing with the dilemma of accessing “what is available” rather than “what we need”?The ending could be stronger. Please consider rewriting this final sentence. Try something like: “Until then, we will keep dealing with the dilemma of accessing “what is available” rather than “what we need”.Response: Change madeFor my personal interest:p.3, column 2, line 26-28 “Some Latin American journals have aimed to lessen the gap by providing the same article, with the same digital object identifier, in more than one language”This is interesting, I would like to learn more. Is this allowed, ethically? Is this not self-plagiarism? How do you cite such publications and do they count as one or two separate works?Response: This is allowed ethically, because it would have the same MEDLINE/PubMed entry. Thus, we do not believe this is self-plagiarism because it does not count as two publications, but just one (same DOI) with two different ways of viewing it. I think this does not pose a big problem, because readers know that it is the same version in different languages. For example, the journal Medwave provides, within the same Pubmed Abstract page two options: English or Spanish such as in this case: https://www.ncbi.nlm.nih.gov/pubmed/26248156"
}
]
},
{
"id": "11881",
"date": "29 Mar 2016",
"name": "Alfonso J. Rodriguez-Morales",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very interesting article facing a challenge for developing countries, costs of publications. Whilst Open Access is excellent in this setting because allows wide access for everybody, also implies costs for authors in most journals, which is also difficult. Particularly this should be considered more in this discussion. Goverments and institutions should be more responsible covering those costs. But also journals should be responsible and consider extreme cases of inability to pay, then offering waivers, and particularly giving different costs for authors from developing countries, regardless of the classification of the World Bank, which is not fair. For example, the HINARI initiative has fallen short for this reason. Many countries there, included Colombia and Peru, were considered according to the WB as countries with an income not classifiable to have access. Then, this is unfair. Anyway, I would like to see more of this discussion in the paper.",
"responses": []
},
{
"id": "12343",
"date": "29 Mar 2016",
"name": "Geoffrey Williams",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is about dreams, and dreams do not always come true, even open access ones.This is an interesting opinion text, and not a researched article. It therefore aims to stimulate discussion about the key issue of open access, particularly in the medical world. It raises the interesting, and often overlooked, issue as to access to research for medical practitioners and not just researchers.There are a number of issues. One is the control of the big publishers who have no intention to release archived texts. Whilst new production may come out as gold open access, there is still a wealth of hidden data. As they say, citing the Finch report, there is a question of cost as publishing one has a cost that the researcher must pay. Another issue is one of language, and that is related to evaluation criteria and indicators. As long as international is seen as a proxy for quality, and as long as the English language will be seen as international, there will be a problem. In years to come, their dream will come increasingly true, but to hasten it a change of mindset on language is vital, especially as Spanish is far from a minority language. The big question will remain archives as the publishers are sitting on a gold mine, and they know it.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-40
|
https://f1000research.com/articles/5-978/v1
|
24 May 16
|
{
"type": "Review",
"title": "Recent advances in metabolic and bariatric surgery",
"authors": [
"Vance L. Albaugh",
"C. Robb Flynn",
"Robyn A. Tamboli",
"Naji N. Abumrad",
"Vance L. Albaugh",
"C. Robb Flynn",
"Robyn A. Tamboli"
],
"abstract": "Obesity and its associated medical conditions continue to increase and add significant burden to patients, as well as health-care systems, worldwide. Bariatric surgery is the most effective treatment for severe obesity and its comorbidities, and resolution of diabetes is weight loss-independent in the case of some operations. Although these weight-independent effects are frequently described clinically, the mechanisms behind them are not well understood and remain an intense area of focus in the growing field of metabolic and bariatric surgery. Perceptions of the mechanisms responsible for the beneficial metabolic effects of metabolic/bariatric operations have shifted from being mostly restrictive and malabsorption over the last 10 to 15 years to being more neuro-hormonal in origin. In this review, we describe recent basic and clinical findings of the major clinical procedures (adjustable gastric banding, vertical sleeve gastrectomy, Roux-en-Y gastric bypass, and biliopancreatic diversion) as well as other experimental procedures (ileal interposition and bile diversion) that recapitulate many of the metabolic effects of these complex operations in a simpler fashion. As the role of bile acids and the gut microbiome on metabolism is becoming increasingly well described, their potential roles in these improvements following metabolic surgery are becoming better appreciated. Bile acid and gut microbiome changes, in light of recent developments, are discussed in the context of these surgical procedures, as well as their implications for future study.",
"keywords": [
"Metabolic surgery",
"Bariatric surgery",
"Morbid obesity",
"adjustable gastric banding",
"vertical sleeve gastrectomy",
"Roux-en-Y gastric bypass",
"biliopancreatic diversion",
"ileal interposition",
"bile diversion"
],
"content": "Introduction\n\nObesity is a significant health-care problem with few treatment options, many of which are only minimally effective in the long term. Medical therapy consisting of intensive lifestyle modification (that is, diet, exercise, and behavioral therapy) fails to maintain significant long-term weight loss. Although medical intervention can lead to modest weight loss in select patients1, 5–10% weight loss in a morbidly obese individual still leaves that patient with significant cardiometabolic risk2,3.\n\nMetabolic and bariatric surgery (in this review, the phrase “metabolic and bariatric surgery” refers to a single entity) is recognized as the most effective treatment for obesity and its associated comorbidities, such as type 2 diabetes4–7, and its usage continues to increase with the increasing prevalence of obesity and metabolic disease. Early studies from Pories and colleagues8 and others9,10 in gastric bypass patients described diabetes resolving almost immediately after surgery. Even though this effect was described more than 30 years ago8,11, its complex underlying mechanisms remain an intense research focus. Multiple reviews and meta-analyses have confirmed a diabetes resolution rate of approximately 80%12–14 and also provide evidence that operating in patients with a body mass index of less than 35 kg/m2 may be warranted as well for non-obese diabetics15–19. Overall, the benefits of bariatric and metabolic surgery continue to be better described, particularly the decreases in cardiovascular disease and cancer mortality20,21. With the alleviation of diabetes and other comorbidities, it is not surprising that bariatric surgery also exhibits cost savings compared with chronic medical treatment of these diseases20,22–24.\n\nOur aim herein is not to focus on the anatomic differences of the particular operations per se but instead to highlight the discoveries and new questions each procedure has provided over about the last 5 years. The field of metabolic and bariatric surgery has a rich history, although our understanding of how these operations lead to their beneficial effects has significantly changed over the last 10 to 15 years. Operations that were originally intended to produce weight loss through combinations of gastric restriction or malabsorption (or both) clearly have metabolic benefits that are independent from either one of these previously long-held beliefs of their mechanism of action. The historical bile diversion and ileal interposition operations are scientifically in vogue once again and are helping to examine the complex role of bile acids in metabolic regulation. We have focused on the insights from the most popular procedures clinically and experimentally, including gastric banding, vertical sleeve gastrectomy (VSG), Roux-en-Y gastric bypass (RYGB), biliopancreatic diversion (BPD), ileal interposition, and bile diversion. Importantly, we have emphasized many of the species-specific changes that must be considered when translating findings to a clinical context. The role of bile acids and the gut microbiome and their potential interaction is discussed. For a comprehensive discussion of the rapidly growing field of metabolic and bariatric surgery, we direct the reader to this excellent review25.\n\n\nPurely restrictive operations\n\nThe contribution of gastric restriction to the efficacy of bariatric surgery is an area that has been well studied clinically and experimentally. The adjustable gastric band (Figure 1) and gastric balloons are two procedures that purely decrease the capacity of the stomach, by either an adjustable external compressive device (that is, the adjustable gastric band) or merely taking up space within the stomach (that is, the gastric balloon). Over the last decade, adjustable gastric banding has continued to fall from its peak clinical usage in 2008 to currently comprising only 10% of bariatric procedures worldwide26. This decline is due to the relative ineffectiveness of banding for long-term weight loss and reduced comorbidity compared to other bariatric procedures. Consistent with this trend and clinical findings, gastric banding has been modeled in rodents27–29 and has been shown to be less effective for long-term weight loss or improvements in glucose tolerance compared with other procedures (for example, RYGB and BPD) that have additional hormonal or malabsorptive characteristics or both greater and more durable effects. In rodent models, gastric banding is purely restrictive and does not confer any additional benefits beyond restriction of food intake29–33. Banding exerts a temporary weight loss that is compensated for over the course of several weeks27. The delayed resolution of diabetes or other metabolic comorbid conditions is greater with, and attributable to, weight loss secondary to decreased food intake and not direct neuro-hormonal effects34,35.\n\nIn this procedure, an external ring is placed around the proximal portion of the stomach and has a balloon that lines the inside portion of the ring. The inflatable balloon is connected to a port in the subcutaneous tissue of the abdomen that allows the balloon volume, and therefore the amount of gastric restriction, to be adjusted.\n\nSimilarly, gastric balloons have recently been approved in the US, although they have been used in Europe for over a decade36. The science behind the balloon is similar to gastric banding in that the balloons are meant to simulate a false sense of gastric distention and promote satiety even after consumption of a minuscule amount of food. Again, similar to the banding procedure, these devices in theory are purely restrictive; however, as they become more popularly used in Europe and the US, further investigation into the potential changes in hormonal or metabolic effects can be studied37,38.\n\n\nMore than gastric restriction: operations with hormonal effects\n\nWithout a doubt the most exciting advancements in the field of metabolic and bariatric surgery over the last decade have been the identification of mechanisms that have challenged the long-held beliefs that “bariatric” surgical procedures induce weight loss purely through a combination of gastric restriction or nutrient malabsorption, or both. Neural, hormonal, and other nutrient signaling pathways that have previously been unrecognized may be mediating many of the metabolic benefits of these surgical procedures. We examine three of these operations to help highlight these novel and alternative mechanisms in the following section.\n\nThe vertical sleeve gastrectomy is a surgical procedure that decreases gastric volume by approximately 70% with excision of a large portion of stomach along the greater curvature (Figure 2). As mentioned above, clinical and experimental evidence has demonstrated that gastric restriction alone is not effective as a long-term solution for obesity or its comorbidities. When VSG was first introduced, it was deemed to be a purely restrictive procedure; however, this view has been transformed on the basis of clinical and experimental observations. With better weight loss and metabolic outcomes compared with gastric banding, VSG has increased in usage over the last decade and become almost as popular as RYGB26. However, whether VSG provides similar remission to obesity and diabetes long-term has yet to be determined. The ongoing STAMPEDE trial (Surgical Therapy And Medications Potentially Eradicate Diabetes Efficiently)4, the first prospective randomized clinical trial designed to compare both VSG and RYGB to intensive lifestyle modification alone, is not designed to make direct comparisons between VSG and RYGB. Regardless, the data from the STAMPEDE trial should give us rough insight into how well VSG compares to intensive lifestyle intervention and medical therapy in the long term.\n\nA majority of the greater curvature is excised in this procedure, creating a tube-like stomach with a marked reduction in gastric capacity.\n\nAside from its clinical usage, VSG has been described in multiple rodent studies and has several interesting physiologic effects that cannot be explained by restriction alone. In fact, data have directly challenged the notion that VSG involves any intrinsic gastric restriction at all. In studies by Grayson and colleagues39, lactating female rats that had previously received a VSG or sham operation were both able to increase their food intake in response to lactation without any evidence of food intake restriction in the VSG rats that were lactating—one of the most energy-demanding processes physiologically when dams routinely double or even triple their food intake40. Initially, VSG was thought to work by excision of the ghrelin-producing portion of the stomach41. Indeed, circulating ghrelin concentrations are significantly decreased up to a year after VSG42. Interestingly, even in the absence of intestinal rearrangement, VSG is associated with increased secretion of the distal intestinal hormones GLP-1 and peptide YY (PYY)43–45. Studies using rodent models of VSG have examined the contribution of these hormonal changes and other mechanisms to VSG efficacy. Mice with genetic deletion of ghrelin or the GLP-1 receptor continue to show weight loss following VSG46,47, suggesting that isolated changes in these hormones cannot explain the efficacy of the procedure. Unlike ghrelin and GLP-1 receptor knockout mice, though, mice deficient in the bile acid receptor FXR (farnesoid X receptor) have completely abrogated effects of VSG while on a high-fat diet48, thus implicating FXR as a major target of VSG. Consistent with this bile acid receptor dependency, VSG is associated with increased plasma bile acid concentrations in the mouse49, but whether the same holds true in humans is not year clear50. Stefater and colleagues25 examined the effects of VSG compared with diet-induced obesity, pair-fed, or chow-fed control rats. In that study, total bile acids were increased by VSG or pair-feeding by unknown mechanisms, which began to approach the higher concentrations of total bile acids reported in the chow-fed controls. How FXR signaling is altered by bariatric surgery and what other pathways may be affected by VSG remain unclear, although a number of other VSG-related effects require further study, including changes to taste preference similar to RYGB as well as changes in intestinal triglyceride metabolism51–54.\n\nThe RYGB (Figure 3) and BPD (Figure 4) operations combine significant intestinal rearrangement with gastric restriction. Each procedure involves creation of a smaller stomach pouch while diverting nutrient flow to varying distal segments of the intestine. The gastric restrictive component is typically less with BPD but diversion of biliopancreatic secretions is more distal, compared with RYGB. Both RYGB and BPD were originally thought to cause weight loss through a combination of malabsorption and gastric restriction. From clinical practice, we know that bariatric surgical patients are at higher risk for certain micronutrient deficiencies, highly suggestive of an intrinsic malabsorptive component leading to weight loss after these procedures55–57. Compared with RYGB patients, BPD patients tend to have more nutritional and gastrointestinal (GI) side effects58, which may indicate a more severe malabsorptive phenotype. Regardless, both operations produce improvements in diabetes/insulin resistance59–62. Several studies have attempted to determine the magnitude of malabsorption following these procedures clinically and experimentally. In terms of macronutrient malabsorption, animal studies suggest a higher degree of malabsorption in RYGB/BPD models, which could contribute to significant weight loss63,64. However, this degree of malabsorption is not consistently observed in practice, and several clinical studies show frank macronutrient malabsorption, minimal macronutrient malabsorption, or no macronutrient malabsorption at all65–68. Both nutrient and macronutrient malabsorption appear to be much more easily observed in the BPD models compared with RYGB65,69. Collectively, macronutrient malabsorption appears to play a much greater role in the case of BPD than RYGB in the weight loss observed in these patients clinically. However, the complex intestinal rearrangement, altered nutrient absorption, and physical separation of biliopancreatic secretions from nutrients in these operations may alter the intestinal nutrient milieu to explain what drives many of the hormonal and histologic changes70 observed in these procedures.\n\nThe stomach is divided, creating a small gastric pouch that is connected through a gastro-jejunostomy to a distal segment of jejunum, which forms the Roux limb of the procedure. The remainder of the stomach is referred to as the “gastric remnant” and drains into the bypassed portion of bowel, referred to as the “biliopancreatic limb”. Bowel continuity is restored for the biliopancreatic limb by a jejuno-jejunostomy that creates the “Y” configuration of the operation. Thus, ingested nutrients proceed rapidly through the stomach pouch and move immediately into the jejunal Roux limb in the absence of bile and pancreatic secretions. Bile and pancreatic secretions drain via the biliopancreatic limb and then mix with the chyme/nutrients at the point of the jejuno-jejunostomy.\n\nThis is a procedure that effectively diverts bile and pancreatic secretions to the distal bowel for mixing with nutrients/chyme, typically much further distal than a Roux-en-Y gastric bypass. This procedure can be performed with or without a partial gastrectomy and is also referred to as a duodenal switch; the “switch” is the diversion of bile and pancreatic secretions from nutrient flow.\n\nGiven the anatomic changes with RYGB and BPD, two opposing hypotheses—referred to as the “foregut hypothesis” and the “hindgut hypothesis”—arose in the field. The foregut hypothesis posits that an unknown factor—neural or hormonal or both—originates from the bypassed intestinal region in RYGB/BPD that promotes insulin resistance. Thus, bypass of that segment is associated with metabolic improvements. There is considerable evidence in support of this hypothesis, proposed as early as the 1980s by Scopinaro and colleagues71, although the identity of such a factor remains elusive72,73. Two studies examined the foregut hypothesis directly in RYGB patients with gastrostomy tubes in the gastric remnant74,75. Hansen and colleagues74 found that reintroduction of nutrients into the excluded foregut in the first 6 weeks after RYGB did not reverse improvement in glucose responses, whereas Pournaras and colleagues75 found reversal between 9 and 24 months after RYGB. Differences between these two studies could be related to early-versus-late post-operative effects or possible enteral feeding prior to testing in the latter study. Animal studies have also had mixed results, showing that duodenal nutrient exclusion is associated with villous hyperplasia but that improvements in glucose tolerance, weight loss, or incretins are mixed and may depend heavily on strain/genetic background76–79. Conversely, the “hindgut hypothesis” posits that the more rapid delivery of nutrients to the hindgut stimulates either neural or hormonal factors (or both) that lead to the metabolic benefits and weight loss. Within the first month after surgery, concomitant increases in postprandial GLP-1 and insulin secretion and an enhanced incretin effect are observed and have positioned GLP-1 as the prime mediator of improved glucose homeostasis after the procedure. Recent data using GLP-1 receptor antagonists have challenged a dominant role for GLP-1. Although these data indicate that the increased GLP-1 contributes to the increased insulin secretion, this does not translate into improved glucose homeostasis80. It is somewhat unfortunate that these opposing theories arose, because the field has come to appreciate that there are likely components of each that could potentially be at work following RYGB and BPD.\n\nAside from foregut bypass, a number of anatomic/histologic changes are associated with the intestinal rearrangement that can be potentially linked to the metabolic changes. Cell proliferation and villous surface area are increased in the Roux limb of RYGB in humans, as are expression levels of genes suggestive of increased energy demands81. A working hypothesis in rats is that Roux limb hypertrophy, secondary to energetic demands on the jejunum, results in increased nutrient uptake following RYGB, making the Roux limb a significant contributor to glucose homeostasis. This is evident by increased basolateral glucose uptake of the Roux limb in the post-absorptive state with corresponding changes in glucose transporters on immunohistologic analysis82. Consistent with this intestinal hypertrophy hypothesis, other procedures have also shown changes in intestinal histology and increased L-cell populations with concomitant changes in L-cell hormonal responses (that is, GLP-1 and PYY)64,83,84. However, histologic changes also occur in the common channel (that is, distal ileum) that could represent hypertrophy secondary to undigested luminal nutrients85.\n\nBesides the improvements in insulin sensitivity/glucose homeostasis and weight loss observed with RYGB, nutrient signaling within the GI tract to alter taste or other metabolic processes is significantly changed by these operations. Several studies have demonstrated altered taste preference for lipid or glucose solutions in humans and animals after RYGB52,86–88. These findings appear to reflect changes in central reward pathways, but whether this reflects direct central effects or actions of peripheral signals needs to be determined88–91. Additionally, similar neural sensing mechanisms operating via the vagus nerve have been implicated in luminal nutrient sensing, which adds another layer of complexity between the neural and hormonal regulation that is changed by RYGB and potentially other bariatric operations. Further studies are needed to identify these mechanisms, as the role of the vagus nerve in these effects remains unclear92–94.\n\nAlthough most of the focus has been placed on the intestines, considerable evidence indicates that negative energy balance may also have beneficial metabolic effects after RYGB. In the immediate post-operative period, significant caloric restriction contributes to the early improvements in glucose metabolism95–97. Within the first month after surgery, improvements in hepatic insulin sensitivity are evident98,99, indicating an important contribution of the liver in mediating the weight loss-independent effects of RYGB. On the other hand, an improvement in peripheral insulin sensitivity, which mediates glucose disposal after a meal, occurs later and is related to the ensuing weight loss99–101. It has been debated whether an increase in energy expenditure is responsible for weight loss post-operatively in these patients. Recent evidence suggests that resting energy expenditure is increased in mice63,102 as well as rats103,104 following RYGB. Unlike animal data, human studies using appropriate methodology (that is, regression modeling105–107) do not support any increases in energy expenditure following RYGB108,109. In fact, the massive weight loss of RYGB occurs in the setting of metabolic adaptation (decreased energy demands greater than expected for the degree of weight loss), suggesting the contribution of hormonal or neuronal factors (or both) in the anorectic effect110. The effects of RYGB on energy expenditure appear to be contrary to those observed in BPD, which Scopinaro and colleagues have reported111. Overall, when examining the metabolic and body weight changes observed both acutely and chronically by RYGB and comparing those with BPD, researchers are tempted to speculate that the BPD operation represents an extreme physiologic response. A response of this magnitude tends to make sense given the apparent effects of BPD on energy expenditure, malabsorption, and long-term body weight, which are more easily observed with that procedure compared with RYGB.\n\n\nExperimental metabolic operations without gastric restriction\n\nThe ileal interposition (Figure 5), also previously referred to as “ileal transposition”, has been an insightful procedure used to examine the mechanisms underlying altered nutrient flow after RYGB or BPD. The procedure involves taking a neurovascular intact segment of near-terminal ileum and interposing it just distal to the ligament of Treitz, effectively producing a model simulating rapid hindgut delivery like RYGB or BPD, but without any gastric restriction. Koopmans and colleagues112 first identified that ileal interposition surprisingly led to decreased food intake in obese rats, which the authors attributed to an “over-stimulated ileum”. In most cases, however, these changes in food intake or body weight are either negligible or modest and this may explain why they are not uniformly detectable—indicating potential importance of genetic background (that is, animal strain) or feeding/housing methods in rodent studies113–116. Moreover, these changes appear to be heavily dependent on the length of the ileal segment interposed; longer interposed segments have more robust metabolic effects that could account for variability of findings117. Consistent with this over-stimulated ileal hypothesis, the ileal interposition has profound effects on glucose homeostasis as well as GI hormone secretion and bile acid metabolism similar to RYGB, BPD, and VSG. Weight-independent improvements in glucose homeostasis in rats are secondary to improved skeletal muscle glucose uptake and insulin signaling via Akt113. These benefits persist in monogenic and polygenic rat models of obesity or diabetes, or both113,114. The interposition procedure is also associated with increased expression and secretion of ileal hormones (that is, GLP-1 and GIP)113,115,116 that correspond to increased enteroendocrine cell numbers118.\n\nA neurovascular intact segment of distal or near-terminal ileum is interposed in the proximal jejunum near the ligament of Treitz. The distal jejunum is then re-anastomosed to the small segment of ileum proximal to the ileocecal valve to re-establish bowel continuity.\n\nAside from ileal hormones, ileal interposition causes marked elevations in circulating bile acids113,116, the mechanism of which remains unclear. However, both basal and nutrient-stimulated bile acid concentrations are a common finding after bariatric operations. In this regard, bile acids are recognized players in the regulation of glucose and lipid metabolism through the FXR and TGR5 receptors, respectively. It is tempting to speculate that activation of those receptors could improve glucose tolerance and enhance insulin sensitivity.\n\nThe mechanisms responsible for the observed elevations in plasma bile acid concentrations following ileal interposition remain unclear. Strader and colleagues119 suggested that the beneficial effects of ileal interposition come from nutrient or bile exposure of the interposed ileal segment at much higher concentrations, thus overwhelming the transposed segment and subsequently causing compensatory changes to the “neo-ileum”. In rats, following ileal interposition the apical sodium bile acid transporter is decreased (~95%) in the interposed ileal segment, although the cytosolic transporter is increased. In those same animals, however, the most distal segments of intestine (that is, the remaining or neo-ileum and/or colon) had robust increases in expression of these transporters119.\n\nBile diversion (Figure 6) was an experimental surgical technique developed in the 1960s120,121 for surgical management of hypercholesterolemia. The proposed mechanism of the procedure was to prevent bile salts from mixing with intestinal contents prior to reaching the colon, and thus cholesterol (and other lipid) absorption would be significantly less. The bile diversion procedure paralleled the development of the jejunal-ileal and ileal bypasses pioneered by Buchwald and Gebhard122 at the University of Minnesota and Scott and colleagues123 at Vanderbilt University, and this culminated in the Program of Surgical Control of Hyperlipidemias (POSCH) trial124. From these studies, as one would expect, there were tremendous improvements in total cholesterol levels that were attributed to the lack of bile-nutrient mixing and thus retarded lipid absorption.\n\nIn the absence of any gastric restriction, the common bile duct is ligated proximal to the pancreatic duct and an anastomosis is created between a portion of ileum and the gallbladder. Pancreatic secretions follow their normal course and drain into the duodenum, but biliary secretions are diverted to the portion of ileum connected directly to the gallbladder.\n\nAt the time bile diversion was developed, bile acids had not yet been recognized as the metabolic hormones that we know today125,126. Thus, more recent studies have revitalized the potential uses for bile diversion as a model to dissect the mechanisms at work following bariatric surgery because of its similarities to other bariatric procedures. Bile diversion, similar to RYGB or BPD, limits bile-nutrient mixing until a more distal point in the intestine. Unlike the RYGB or BPD, though, there is no biliopancreatic limb for these secretions to flow; they are completely diverted by cannula or anastomosis directly to the intestinal segment of interest maintaining unaltered alimentary flow. Coincidentally, it was noted that glucose tolerance was improved even in the absence of weight loss in dogs after bile diversion127. Manfredini and colleagues128 replicated these observations and described improvements in oral and intravenous glucose tolerance in the absence of any changes in insulin secretion. However, a recent report of bile diversion in lean rats has shown conflicting results on whether insulin secretion is altered, and this requires further study128,129.\n\nRegardless of whether insulin secretion is altered, there is improved insulin responsiveness and decreased fasting glucose after bile diversion suggestive of a change in insulin sensitivity in lean rats129. Similarly, in a high fat-fed model, Kohli and colleagues130 have shown that bile diversion to the jejunum produces weight loss compared with sham/control rats. These beneficial changes are recapitulated with oral bile acid administration and appear to be mediated by alleviating endoplasmic reticulum stress. Moreover, bile diversion in rats was associated with increased number and length of villi, similar to procedures like RYGB, BPD, and ileal interposition130,131. Each of the bile diversion models is associated with increased circulating bile acid concentrations, which appear to be necessary for the metabolic effects since administration of bile acid sequestrants (for example, cholestyramine) normalizes plasma bile acid concentrations and abolishes the metabolic improvements. These findings are consistent with our recent studies using a gallbladder to ileum anastomosis in the mouse, which compared it alongside RYGB in a mouse model of diet-induced obesity. Similar to the findings in rats, bile diversion in mice is associated with striking improvements in glucose tolerance, insulin sensitivity, normalization of blood lipids, and complete resolution of hepatic steatosis63. These changes are secondary to a degree of malabsorption of dietary lipid in the mice; however, they are also observed with marked increases in total circulating bile acids—specific bile acids that are implicated in metabolic signaling through bile acids receptors (that is, TGR5 and FXR)125,132.\n\n\nRole of bile acids and the gut microbiome\n\nThe contributions of bile acids in the metabolic effects following bariatric surgery are at the forefront of current investigation. There is considerable evidence that bile acid concentrations are increased following bariatric surgical procedures—both clinical and experimental—and the most robust clinical changes are observed in RYGB and BPD133–138. Whether the changes in circulating bile acid concentrations lead to changes in known downstream metabolic effectors like fibroblast growth factor 19 (FGF19, FGF15 in rodents) is still under investigation139. The changes in bile acid metabolism and circulating concentrations appear to be dynamic following RYGB133 and this may explain why some investigators observe increases in the metabolically beneficial hormone FGF19 and some do not133,138,140–142. From a mechanistic perspective, the fact that the ileal interposition and bile diversion procedures recapitulate these bile acid elevations is quite intriguing and implies that either absence of bile in the proximal intestine or overabundance in the distal intestine may contribute the metabolic effects observed. The changes in bile acids appear to be mediated in part by increases in bile acid synthesis through increased expression of CYP7A1, the rate-limiting enzyme in bile acid synthesis. However, there are also concurrent changes in bile acid transporter proteins in the liver and the ileum63 that would also be expected to increase the circulating total bile acid pool. Paradoxically, bile acid synthesis is elevated in the face of increased SHP and decreased FXR expression in the livers of these animals. These changes in SHP and FXR expression are inconsistent with our current understanding of hepatic bile acid synthesis143. Regardless of how bile acid concentrations are increased following the ileal interposition and bile diversion procedures, the molecular mechanisms of how the increased abundance of bile acids may alter glucose homeostasis is currently unknown. Evidence suggests that these effects are mediated via FXR or TGR5 signaling (or both) in the beta-cell or in the enterocytes themselves (or in both)125,144,145, but further studies need to focus on clarifying these mechanisms and their physiologic importance. These bile acid-mediated effects on glucose and lipid metabolism have therapeutic implications on diabetes irrespectively of changes on body weight and are a focus of current investigation.\n\nPerhaps one of the most intriguing observations of this century thus far has been that fecal transfer of gut microbiota from obese donors causes weight gain in lean recipients146. Similarly, the opposite effect can be demonstrated in obese mice with stool from RYGB mouse donors, which demonstrates the transferability of at least some of the metabolic benefits of gastric bypass147. There has been significant evidence that the gut microbiota is altered following bariatric surgery, although the mechanisms of these changes and the potential contribution they make in the metabolic benefits post-operatively are unknown. Changes in the microbiota have been examined in mouse models of sleeve gastrectomy48 as well as biliary diversion63, and these procedures show what appear to be beneficial changes. Interestingly, le Roux and colleagues148 have recently shown that the gut microbiota alter the pattern of adipose tissue deposition. The interaction between the gut microbiota and bile acid metabolism is complex, and particular bile acids are antibacterial and likely affect the gut microbiota. In contrast, the gut microbiota are the major source of bile acid diversity, chemically transforming endogenously produced bile acids to a number of different chemical species that likely have varying potencies at bile acid receptors. At this time, it is not clear whether these changes in microbiota are a cause or an effect of the metabolic improvements and weight loss observed following bariatric surgery, but the potential for altering the microbiome as a treatment for obesity or diabetes (or both) continues to emerge149.\n\n\nSummary and future directions\n\nFrom a clinical perspective, prospective clinical trials are needed to directly compare not only durability of weight loss but also resolution of other obesity comorbidities (for example, diabetes, insulin resistance, and hyperlipidemias) after VSG and RYGB150. Scientifically, while much progress has been made regarding the weight loss-independent metabolic effects of bariatric surgery, the complexity of the system continues to provide challenges. The field is progressing from a “GLP-1-centric view” of bariatric surgery to encompass the importance of early benefits of caloric restriction and non-traditional regulators of metabolism. The changes in bile acid metabolism with bariatric surgery will continue to drive the study of these procedures and the mechanisms in animal models—especially as the bile acid field continues to advance our understanding of these potent metabolic regulators. Lastly, the interaction of the bile acid milieu and the gut microbiome cannot be ignored. The gut microbiome field continues to grow and become more intervention-driven, as opposed to descriptive, and will continue to help identify the changes underlying the metabolic benefits of bariatric and metabolic surgery. These continue to be exciting times for metabolic and bariatric surgical research, and future studies examining the contributions of bile acids and microbiota both clinically and experimentally may lead to more effective treatments, and perhaps new interventional procedures for obesity and diabetes.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe National Institute of Diabetes and Digestive and Kidney Diseases supported this work through the following grants: F32DK103474 (VLA), R01DK091748, R01DK070860, R01 DK105847, R01DK100431, R24DK093421-01A1, and DK058404 (Vanderbilt Digestive Disease Research Center), DK20593 (Vanderbilt Diabetes Research and Training Center), and 1UL1RR024975 (Vanderbilt Clinical and Translational Science Award).\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\nThe authors would like to thank Henry Buchwald (University of Minnesota) for his historical insight into the ileal bypass and early bile diversion procedures.\n\n\nReferences\n\nBlackburn G: Effect of degree of weight loss on health benefits. Obes Res. 1995; 3(Suppl 2): 211s–216s. 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Am J Surg. 2007; 194(2): 221–4. PubMed Abstract | Publisher Full Text\n\nRubino F, Marescaux J: Effect of duodenal-jejunal exclusion in a non-obese animal model of type 2 diabetes: a new perspective for an old disease. Ann Surg. 2004; 239(1): 1–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHansen EN, Tamboli RA, Isbell JM, et al.: Role of the foregut in the early improvement in glucose tolerance and insulin sensitivity following Roux-en-Y gastric bypass surgery. Am J Physiol Gastrointest Liver Physiol. 2011; 300(5): G795–802. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPournaras DJ, Aasheim ET, Bueter M, et al.: Effect of bypassing the proximal gut on gut hormones involved with glycemic control and weight loss. Surg Obes Relat Dis. 2012; 8(4): 371–4. PubMed Abstract | Publisher Full Text\n\nHabegger KM, Al-Massadi O, Heppner KM, et al.: Duodenal nutrient exclusion improves metabolic syndrome and stimulates villus hyperplasia. Gut. 2014; 63(8): 1238–46. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKindel TL, Martins PJ, Yoder SM, et al.: Bypassing the duodenum does not improve insulin resistance associated with diet-induced obesity in rodents. Obesity (Silver Spring). 2011; 19(2): 380–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKindel TL, Yoder SM, Seeley RJ, et al.: Duodenal-jejunal exclusion improves glucose tolerance in the diabetic, Goto-Kakizaki rat by a GLP-1 receptor-mediated mechanism. J Gastrointest Surg. 2009; 13(10): 1762–72. PubMed Abstract | Publisher Full Text\n\nRubino F, Forgione A, Cummings DE, et al.: The mechanism of diabetes control after gastrointestinal bypass surgery reveals a role of the proximal small intestine in the pathophysiology of type 2 diabetes. Ann Surg. 2006; 244(5): 741–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVetter ML, Wadden TA, Teff KL, et al.: GLP-1 plays a limited role in improved glycemia shortly after Roux-en-Y gastric bypass: a comparison with intensive lifestyle modification. Diabetes. 2015; 64(2): 434–46. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSpak E, Björklund P, Helander HF, et al.: Changes in the mucosa of the Roux-limb after gastric bypass surgery. Histopathology. 2010; 57(5): 680–8. PubMed Abstract | Publisher Full Text\n\nSaeidi N, Meoli L, Nestoridi E, et al.: Reprogramming of intestinal glucose metabolism and glycemic control in rats after gastric bypass. Science. 2013; 341(6144): 406–10. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBorg CM, le Roux CW, Ghatei MA, et al.: Biliopancreatic diversion in rats is associated with intestinal hypertrophy and with increased GLP-1, GLP-2 and PYY levels. Obes Surg. 2007; 17(9): 1193–8. PubMed Abstract | Publisher Full Text\n\nRhee NA, Wahlgren CD, Pedersen J, et al.: Effect of Roux-en-Y gastric bypass on the distribution and hormone expression of small-intestinal enteroendocrine cells in obese patients with type 2 diabetes. Diabetologia. 2015; 58(10): 2254–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nle Roux CW, Borg C, Wallis K, et al.: Gut hypertrophy after gastric bypass is associated with increased glucagon-like peptide 2 and intestinal crypt cell proliferation. Ann Surg. 2010; 252(1): 50–6. PubMed Abstract | Publisher Full Text\n\nPepino MY, Bradley D, Eagon JC, et al.: Changes in taste perception and eating behavior after bariatric surgery-induced weight loss in women. Obesity (Silver Spring). 2014; 22(5): E13–20. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPepino MY, Stein RI, Eagon JC, et al.: Bariatric surgery-induced weight loss causes remission of food addiction in extreme obesity. Obesity (Silver Spring). 2014; 22(8): 1792–8. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHajnal A, Kovacs P, Ahmed T, et al.: Gastric bypass surgery alters behavioral and neural taste functions for sweet taste in obese rats. Am J Physiol Gastrointest Liver Physiol. 2010; 299(4): G967–79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThanos PK, Subrize M, Delis F, et al.: Gastric bypass increases ethanol and water consumption in diet-induced obese rats. Obes Surg. 2012; 22(12): 1884–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDunn JP, Cowan RL, Volkow ND, et al.: Decreased dopamine type 2 receptor availability after bariatric surgery: preliminary findings. Brain Res. 2010; 1350: 123–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThanos PK, Michaelides M, Subrize M, et al.: Roux-en-Y Gastric Bypass Alters Brain Activity in Regions that Underlie Reward and Taste Perception. PLoS One. 2015; 10(6): e0125570. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPal A, Rhoads DB, Tavakkoli A: Foregut exclusion disrupts intestinal glucose sensing and alters portal nutrient and hormonal milieu. Diabetes. 2015; 64(6): 1941–50. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nShin AC, Zheng H, Berthoud HR: Vagal innervation of the hepatic portal vein and liver is not necessary for Roux-en-Y gastric bypass surgery-induced hypophagia, weight loss, and hypermetabolism. Ann Surg. 2012; 255(2): 294–301. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBhutta HY, Rajpal N, White W, et al.: Effect of Roux-en-Y gastric bypass surgery on bile acid metabolism in normal and obese diabetic rats. PLoS One. 2015; 10(3): e0122273. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nIsbell JM, Tamboli RA, Hansen EN, et al.: The importance of caloric restriction in the early improvements in insulin sensitivity after Roux-en-Y gastric bypass surgery. Diabetes Care. 2010; 33(7): 1438–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJackness C, Karmally W, Febres G, et al.: Very low-calorie diet mimics the early beneficial effect of Roux-en-Y gastric bypass on insulin sensitivity and β-cell Function in type 2 diabetic patients. Diabetes. 2013; 62(9): 3027–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOliván B, Teixeira J, Bose M, et al.: Effect of weight loss by diet or gastric bypass surgery on peptide YY3-36 levels. Ann Surg. 2009; 249(6): 948–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDunn JP, Abumrad NN, Breitman I, et al.: Hepatic and peripheral insulin sensitivity and diabetes remission at 1 month after Roux-en-Y gastric bypass surgery in patients randomized to omentectomy. Diabetes Care. 2012; 35(1): 137–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBojsen-Møller KN, Dirksen C, Jørgensen NB, et al.: Early enhancements of hepatic and later of peripheral insulin sensitivity combined with increased postprandial insulin secretion contribute to improved glycemic control after Roux-en-Y gastric bypass. Diabetes. 2014; 63(5): 1725–37. PubMed Abstract | Publisher Full Text\n\nTamboli RA, Breitman I, Marks-Shulman PA, et al.: Early weight regain after gastric bypass does not affect insulin sensitivity but is associated with elevated ghrelin. Obesity (Silver Spring). 2014; 22(7): 1617–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCampos GM, Rabl C, Peeva S, et al.: Improvement in peripheral glucose uptake after gastric bypass surgery is observed only after substantial weight loss has occurred and correlates with the magnitude of weight lost. J Gastrointest Surg. 2010; 14(1): 15–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNestoridi E, Kvas S, Kucharczyk J, et al.: Resting energy expenditure and energetic cost of feeding are augmented after Roux-en-Y gastric bypass in obese mice. Endocrinology. 2012; 153(5): 2234–44. PubMed Abstract | Publisher Full Text\n\nStylopoulos N, Hoppin AG, Kaplan LM: Roux-en-Y gastric bypass enhances energy expenditure and extends lifespan in diet-induced obese rats. Obesity (Silver Spring). 2009; 17(10): 1839–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShin AC, Zheng H, Townsend RL, et al.: Longitudinal assessment of food intake, fecal energy loss, and energy expenditure after Roux-en-Y gastric bypass surgery in high-fat-fed obese rats. Obes Surg. 2013; 23(4): 531–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaiyala KJ: Mathematical model for the contribution of individual organs to non-zero y-intercepts in single and multi-compartment linear models of whole-body energy expenditure. PLoS One. 2014; 9(7): e103301. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTschöp MH, Speakman JR, Arch JR, et al.: A guide to analysis of mouse energy metabolism. Nat Methods. 2011; 9(1): 57–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nButler AA, Kozak LP: A recurring problem with the analysis of energy expenditure in genetic models expressing lean and obese phenotypes. Diabetes. 2010; 59(2): 323–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchmidt JB, Pedersen SD, Gregersen NT, et al.: Effects of RYGB on energy expenditure, appetite and glycaemic control: a randomized controlled clinical trial. Int J Obes (Lond). 2016; 40(2): 281–90. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nTamboli RA, Hossain HA, Marks PA, et al.: Body composition and energy metabolism following Roux-en-Y gastric bypass surgery. Obesity (Silver Spring). 2010; 18(9): 1718–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnuth ND, Johannsen DL, Tamboli RA, et al.: Metabolic adaptation following massive weight loss is related to the degree of energy imbalance and changes in circulating leptin. Obesity (Silver Spring). 2014; 22(12): 2563–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdami GF, Compostano A, Bessarione D, et al.: Resting Energy Expenditure in Long-Term Postobese Subjects after Weight Normalization by Dieting or Biliopancreatic Diversion. Obes Surg. 1993; 3(4): 397–9. PubMed Abstract | Publisher Full Text\n\nKoopmans HS, Sclafani A, Fichtner C, et al.: The effects of ileal transposition on food intake and body weight loss in VMH-obese rats. Am J Clin Nutr. 1982; 35(2): 284–93. PubMed Abstract\n\nCulnan DM, Albaugh V, Sun M, et al.: Ileal interposition improves glucose tolerance and insulin sensitivity in the obese Zucker rat. Am J Physiol Gastrointest Liver Physiol. 2010; 299(3): G751–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCummings BP, Strader AD, Stanhope KL, et al.: Ileal interposition surgery improves glucose and lipid metabolism and delays diabetes onset in the UCD-T2DM rat. Gastroenterology. 2010; 138(7): 2437–46, 2446.e1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStrader AD, Vahl TP, Jandacek RJ, et al.: Weight loss through ileal transposition is accompanied by increased ileal hormone secretion and synthesis in rats. Am J Physiol Endocrinol Metab. 2005; 288(2): E447–53. PubMed Abstract | Publisher Full Text\n\nStrader AD, Clausen TR, Goodin SZ, et al.: Ileal interposition improves glucose tolerance in low dose streptozotocin-treated diabetic and euglycemic rats. Obes Surg. 2009; 19(1): 96–104. PubMed Abstract | Publisher Full Text\n\nRamzy AR, Nausheen S, Chelikani PK: Ileal transposition surgery produces ileal length-dependent changes in food intake, body weight, gut hormones and glucose metabolism in rats. Int J Obes (Lond). 2014; 38(3): 379–87. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHansen CF, Vassiliadis E, Vrang N, et al.: The effect of ileal interposition surgery on enteroendocrine cell numbers in the UC Davis type 2 diabetes mellitus rat. Regul Pept. 2014; 189: 31–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nTam CS, Berthoud HR, Bueter M, et al.: Could the mechanisms of bariatric surgery hold the key for novel therapies? report from a Pennington Scientific Symposium. Obes Rev. 2011; 12(11): 984–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKeig HE, Belmonte RL Jr, McKain JM, et al.: The effect of bile diversion on induced hypercholesterolemia. Arch Surg. 1968; 96(4): 613–8. PubMed Abstract\n\nMorgan CV Jr, Lanier VC, Finch WT, et al.: Protective effects of bile diversion to the distal fourth of small intestine against experimental hypercholesterolemia and atherosclerosis in dogs. Am Surg. 1972; 38(1): 10–2. PubMed Abstract\n\nBuchwald H, Gebhard RL: Effect of Intestinal Bypass on Cholesterol Absorption and Blood Levels in the Rabbit. Am J Physiol. 1964; 207: 567–72. PubMed Abstract\n\nScott HW Jr, Stephenson SE Jr, Younger R, et al.: Prevention of experimental atherosclerosis by ileal bypass: 20 per cent cholesterol diet and I-131 induced hypothyroidism in dogs. Ann Surg. 1966; 163(5): 795–807. PubMed Abstract | Free Full Text\n\nBuchwald H, Varco RL, Matts JP, et al.: Effect of partial ileal bypass surgery on mortality and morbidity from coronary heart disease in patients with hypercholesterolemia. Report of the Program on the Surgical Control of the Hyperlipidemias (POSCH). N Engl J Med. 1990; 323(14): 946–55. PubMed Abstract | Publisher Full Text\n\nThomas C, Gioiello A, Noriega L, et al.: TGR5-mediated bile acid sensing controls glucose homeostasis. Cell Metab. 2009; 10(3): 167–77. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLefebvre P, Cariou B, Lien F, et al.: Role of bile acids and bile acid receptors in metabolic regulation. Physiol Rev. 2009; 89(1): 147–91. PubMed Abstract | Publisher Full Text\n\nLeriche R, Joung A: Essai sur le traitement chirurgical du diabete par la derivation biliaire: documents experimentaux et cliniques. Annales dEndocrinologie. 1939; 1: 3–10.\n\nManfredini G, Ermini M, Scopsi L, et al.: Internal biliary diversion improves glucose tolerance in the rat. Am J Physiol. 1985; 249(4 Pt 1): G519–27. PubMed Abstract\n\nGoncalves D, Barataud A, De Vadder F, et al.: Bile Routing Modification Reproduces Key Features of Gastric Bypass in Rat. Ann Surg. 2015; 262(6): 1006–15. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKohli R, Setchell KD, Kirby M, et al.: A surgical model in male obese rats uncovers protective effects of bile acids post-bariatric surgery. Endocrinology. 2013; 154(7): 2341–51. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKohli R, Kirby M, Setchell KD, et al.: Intestinal adaptation after ileal interposition surgery increases bile acid recycling and protects against obesity-related comorbidities. Am J Physiol Gastrointest Liver Physiol. 2010; 299(3): G652–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSayin SI, Wahlström A, Felin J, et al.: Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist. Cell Metab. 2013; 17(2): 225–35. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAlbaugh VL, Flynn CR, Cai S, et al.: Early Increases in Bile Acids Post Roux-en-Y Gastric Bypass Are Driven by Insulin-Sensitizing, Secondary Bile Acids. J Clin Endocrinol Metab. 2015; 100(9): E1225–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSteinert RE, Peterli R, Keller S, et al.: Bile acids and gut peptide secretion after bariatric surgery: a 1-year prospective randomized pilot trial. Obesity (Silver Spring). 2013; 21(12): E660–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWerling M, Vincent RP, Cross GF, et al.: Enhanced fasting and post-prandial plasma bile acid responses after Roux-en-Y gastric bypass surgery. Scand J Gastroenterol. 2013; 48(11): 1257–64. PubMed Abstract | Publisher Full Text\n\nPatti ME, Houten SM, Bianco AC, et al.: Serum bile acids are higher in humans with prior gastric bypass: potential contribution to improved glucose and lipid metabolism. Obesity (Silver Spring). 2009; 17(9): 1671–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDutia R, Embrey M, O'Brien S, et al.: Temporal changes in bile acid levels and 12α-hydroxylation after Roux-en-Y gastric bypass surgery in type 2 diabetes. Int J Obes (Lond). 2015; 39(5): 806–13. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nFerrannini E, Camastra S, Astiarraga B, et al.: Increased Bile Acid Synthesis and Deconjugation After Biliopancreatic Diversion. Diabetes. 2015; 64(10): 3377–85. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRyan KK, Kohli R, Gutierrez-Aguilar R, et al.: Fibroblast growth factor-19 action in the brain reduces food intake and body weight and improves glucose tolerance in male rats. Endocrinology. 2013; 154(1): 9–15. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPournaras DJ, Glicksman C, Vincent RP, et al.: The role of bile after Roux-en-Y gastric bypass in promoting weight loss and improving glycaemic control. Endocrinology. 2012; 153(8): 3613–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGerhard GS, Styer AM, Wood GC, et al.: A role for fibroblast growth factor 19 and bile acids in diabetes remission after Roux-en-Y gastric bypass. Diabetes Care. 2013; 36(7): 1859–64. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nJørgensen NB, Dirksen C, Bojsen-Møller KN, et al.: Improvements in glucose metabolism early after gastric bypass surgery are not explained by increases in total bile acids and fibroblast growth factor 19 concentrations. J Clin Endocrinol Metab. 2015; 100(3): E396–406. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLi S, Hsu DD, Li B, et al.: Cytoplasmic tyrosine phosphatase Shp2 coordinates hepatic regulation of bile acid and FGF15/19 signaling to repress bile acid synthesis. Cell Metab. 2014; 20(2): 320–32. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDüfer M, Hörth K, Wagner R, et al.: Bile acids acutely stimulate insulin secretion of mouse β-cells via farnesoid X receptor activation and KATP channel inhibition. Diabetes. 2012; 61(6): 1479–89. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTrabelsi MS, Daoudi M, Prawitt J, et al.: Farnesoid X receptor inhibits glucagon-like peptide-1 production by enteroendocrine L cells. Nat Commun. 2015; 6: 7629. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBäckhed F, Ding H, Wang T, et al.: The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci U S A. 2004; 101(44): 15718–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiou AP, Paziuk M, Luevano JM Jr, et al.: Conserved shifts in the gut microbiota due to gastric bypass reduce host weight and adiposity. Sci Transl Med. 2013; 5(178): 178ra41. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTremaroli V, Karlsson F, Werling M, et al.: Roux-en-Y Gastric Bypass and Vertical Banded Gastroplasty Induce Long-Term Changes on the Human Gut Microbiome Contributing to Fat Mass Regulation. Cell Metab. 2015; 22(2): 228–38. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nChen Z, Guo L, Zhang Y, et al.: Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. J Clin Invest. 2014; 124(8): 3391–406. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nVidal J, Jiménez A, de Hollanda A, et al.: Metabolic Surgery in Type 2 Diabetes: Roux-en-Y Gastric Bypass or Sleeve Gastrectomy as Procedure of Choice? Curr Atheroscler Rep. 2015; 17(10): 58. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13421",
"date": "24 May 2016",
"name": "Alfonso Torquati",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13422",
"date": "24 May 2016",
"name": "Mario Musella",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-978
|
https://f1000research.com/articles/5-607/v1
|
08 Apr 16
|
{
"type": "Case Report",
"title": "Case Report: Multifocal biphasic squamoid alveolar renal cell carcinoma",
"authors": [
"José I. López"
],
"abstract": "A multifocal biphasic squamoid alveolar renal cell carcinoma in a 68-year-old man is reported. Four different peripheral tumor nodules were identified on gross examination. A fifth central tumor corresponded to a conventional clear cell renal cell carcinoma. Biphasic squamoid alveolar renal cell carcinoma is a rare tumor that has been very recently characterized as a distinct histotype within the spectrum of papillary renal cell carcinoma. Immunostaining with cyclin D1 seems to be specific of this tumor subtype. This is the first reported case with multifocal presentation.",
"keywords": [
"Kidney",
"biphasic squamoid alveolar renal cell carcinoma",
"papillary renal cell carcinoma",
"immunohistochemistry"
],
"content": "Introduction\n\nThe so-called biphasic squamoid alveolar renal cell carcinoma (BSARCC) was described for the first time in 2012 by Petersson et al.1 and has been very recently revisited and fully characterized by Hes et al.2. Histological, immunohistochemical, comparative genomic hybridization and fluorescence in situ hybridization analyses have revealed that BSARCC is a renal neoplasm closely related to papillary renal cell carcinoma (PRCC)2.\n\nThe present paper describes a new BSARCC with multifocal presentation that was associated with a conventional clear cell renal cell carcinoma (CCRCC). To note, multifocality has not been reported in BSARCC so far.\n\n\nCase report\n\nA 68-year-old man presented with transient hematuria. CT scan revealed multiple tumors on his right kidney, four of them being located at the periphery (Figure 1). Radical nephrectomy was performed. The post-surgery period did not show any clinical complications. The patient was asymptomatic and free of disease at the last contact, 6 months after diagnosis.\n\nCT scans show multiple tumors in the right kidney (A and B). Gross examination displays a yellowish central tumor with solid-cystic areas corresponding to a clear cell renal cell carcinoma (C) and four peripheral whitish tumors and several intrarenal micronodules corresponding to biphasic squamoid alveolar renal cell carcinomas (C and D).\n\nOn gross examination up to five tumors and several small intrarenal micronodules were discovered (Figure 1). Four tumors were subcapsular and showed a whitish homogeneous cut surface, measuring between 1 and 3 cm in diameter. The fifth tumor was centrally located, presented mixed solid and cystic areas with a yellowish cut surface and measured 4.5 cm in diameter.\n\nHistologically, the yellowish central tumor was a conventional organ-confined CCRCC grade 1 (ISUP 2013)3. On low-power view, all the whitish peripheral tumors and the micronodules displayed a similar histology consisting in areas reminiscent to glomerular-like structures (Figure 2 and Figure 3) alternating with others typical of type 1 PRCC. On high magnification, these structures were composed of a single row of small cells with scant cytoplasm displaying an alveolar disposition. The alveoli were filled with cell groups with large cytoplasm and squamoid appearance. True squamous cell differentiation, however, was not observed. Mitosis and necrosis were not seen.\n\nPanoramic view of one of the biphasic squamoid alveolar renal cell carcinomas displaying a conventional type 1 papillary renal cell carcinoma component (A). Alveolar structures filled with large cells are typical of biphasic squamoid alveolar renal cell carcinoma (B).\n\nBy immunohistochemistry, the tumor was positive with CK7, vimentin, PAX-8, racemase, RCC marker, AE1/AE3, 34βE12, carbonic anhydrase IX, CD10, and cyclin D1. Immunostaining pattern was distinct depending on the cell type. For instance, cyclin D1 and 34βE12 immunostained selectively the squamoid cells whilst RCC marker and carbonic anhydrase IX did it only in small alveoli-forming cells. The rest of the antibodies immunostained both cell types. The tumor was negative with p63 and CK20.\n\n\nDiscussion\n\nBSARCC is a recently recognized variant of renal carcinoma1,2. Its pathological diagnosis can be suggested on hematoxylin-eosin slides and is based on the recognition of two different cell types arranged in a distinct architecture. Small groups of large cells with abundant cytoplasm and squamoid appearance are surrounded by small cells with scant cytoplasm forming alveolar-like structures. This distinct growth pattern can be more or less evident in different tumor areas and, same as happens in the case here presented, can be combined with areas of conventional PRCC2. The combination of BSARCC and PRCC histologies in almost half of the previously published cases favors the inclusion of this tumor within the broad spectrum of PRCC2. No association of BSARCC with CCRCC, as in the case here presented, has been reported so far.\n\nMorphological diagnostic features of BSARCC can be supported by immunohistochemistry and, if necessary, by genetics. All BSARCC reported to date are positive with cytokeratin 7, epithelial membrane antigen, vimentin and, interestingly, cyclin D1. Cyclin D1 immunostaining is specific of this tumor and helps in its recognition in daily practice. Interestingly, Cyclin D1 immunostaining is restricted to the centrally located squamoid cells. Molecular-genetic data show gains of chromosomes 7 and 17, thus linking BSARCC to PRCC.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for publication of this case report and any accompanying images and/or other details that could potentially reveal the patient’s identity.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPetersson F, Bulimbasic S, Hes O, et al.: Biphasic alveolosquamoid renal carcinoma: a histomorphological, immunohistochemical, molecular genetic, and ultrastructural study of a distinctive morphologic variant of renal cell carcinoma. Ann Diagn Pathol. 2012; 16(6): 459–469. PubMed Abstract | Publisher Full Text\n\nHes O, Condom Mundo E, Peckova K, et al.: Biphasic squamoid alveolar renal cell carcinoma: A distinctive subtype of papillary renal cell carcinoma? Am J Surg Pathol. 2016. PubMed Abstract | Publisher Full Text\n\nDelahunt B, Cheville JC, Martignoni G, et al.: The International Society of Urological Pathology (ISUP) grading system for renal cell carcinoma and other prognostic parameters. Am J Surg Pathol. 2013; 37(10): 1490–1504. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13307",
"date": "20 Apr 2016",
"name": "Guido Martignoni",
"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 describes a multifocal biphasic squamoid alveolar renal cell carcinoma in association of clear cell renal cell carcinoma. The Author claims that staining for cyclin D1 is specific for this entity; however, labelling for cyclin D1 has been reported in clear cell renal cell carcinoma, papillary renal cell carcinoma, chromophobe renal cell carcinoma and oncocytoma (1, 2) and more recently in clear cell papillary renal cell carcinoma (3) and SDH-deficient renal cell carcinoma (4). Since the immunoreactivity might depend of the clone used, the Author should supply information regarding the antibody to cyclin D1. It would be also interesting to know whether cyclin D1 is positive in the clear cell renal cell carcinoma. Finally, pictures of clear cell renal carcinoma and more detailed pictures demonstrating the squamoid features of biphasic squamoid alveolar renal cell carcinoma should be added.",
"responses": []
},
{
"id": "13402",
"date": "28 Apr 2016",
"name": "Jose Luis Rodriguez-Peralto",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 for the first time a multifocal variant of the recently described squamoid alveolar renal cell carcinoma, a distinct microscopic tumor in the spectrum of papillary renal cell carcinoma.. This is the first reported case on this location. The paper is original, elegant and well written, and the quality of figures nicely reflect the morphologic, and immunohistochemical aspects of this infrequent neoplasm. The references are acceptable and recent. Thus, I consider this paper acceptable in the current version, but some changes may improve the content of the manuscript:Since Cyclin D1 has also been reported in other variant of renal cell carcinoma, I recommend to the author to delete in the discussion the sentence Cyclin D1 immunohistochemistry is specific of this tumor. Moreover, I also included a sentence emphasizing that although Cyclin D1 is characteristic of BSARCC, it may be rarely observed in other variants of RCC. I considered that there are many immunohistochemical pictures of Cyclin D1, I would change figure C by a new H&E of the conventional clear cell renal cell carcinoma",
"responses": []
},
{
"id": "13306",
"date": "28 Apr 2016",
"name": "Claudio Doglioni",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 describes an unusual multifocal presentation of Biphasic squanoid alveolar renal carcinoma associated with a conventional Clear Cell Carcinoma. BSARCC is a recently described rare entity with a peculiar squamoid differentiation characterized by cyclin D1 immunoreactivity; Cyclin D1 per se is not limited to this type of renal tumor, but its pattern of reactivity seems specific for BSARCC. I suggest, considering some, although limited, similarity to squamoid morules in other epithelial tumors (colon, endometrium) to perform also immunostaining for beta-catenin.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-607
|
https://f1000research.com/articles/5-976/v1
|
24 May 16
|
{
"type": "Study Protocol",
"title": "Assessing change in patient-reported quality of life after elective surgery: protocol for an observational comparison study",
"authors": [
"Vanessa L. Kronzer",
"Michelle R. Jerry",
"Michael S. Avidan",
"Vanessa L. Kronzer",
"Michelle R. Jerry"
],
"abstract": "Despite their widespread use, the two main methods of assessing quality of life after surgery have never been directly compared. To support patient decision-making and study design, we aim to compare these two methods. The first of these methods is to assess quality of life before surgery and again after surgery using the same validated scale. The second is simply to ask patients whether or not they think their post-operative quality of life is better, worse, or the same. Our primary objective is to assess agreement between the two measures. Secondary objectives are to calculate the minimum clinically important difference (MCID) and to describe the variation across surgical specialties. To accomplish these aims, we will administer surveys to patients undergoing elective surgery, both before surgery and again 30 days after surgery. This protocol follows detailed guidelines for observational study protocols.",
"keywords": [
"quality of life",
"surgical procedures",
"outcome assessment",
"postoperative period"
],
"content": "Background\n\nThe following protocol follows published guidelines for observational study protocols1. The research question for this study is how validated measures compare to self-reported measures of quality of life in patients undergoing elective surgery. To answer this question, we performed a literature search in PubMed.\n\nResearch studying patient-reported quality of life is burgeoning, including quality of life related to surgery2,3. Two main methods are used to estimate a procedure’s impact on quality of life. The first is to compare patient scores before and after surgery using a validated quality of life scale4,5. The second method is to ask patients about their change in quality of life after surgery occurs6–8. This type of self-reported “global measure” is growing in popularity9. However, patients’ perceived change in quality of life may be inaccurate due to cognitive biases such as choice-supportive bias10,11, or theory-driven recall bias12,13. Uncovering potential bias in measures of quality of life is important since patients and clinicians base their surgical decision-making on these measures.\n\nIn addition, the minimum clinically important difference (MCID) in quality of life has been established in the literature for general populations14,15 and for neurosurgical populations16,17, but has not been studied in general surgical populations. Establishing a MCID for quality of life is crucial, both for patients deciding whether or not to receive surgery and for clinicians evaluating the effectiveness of surgery.\n\nWith its large sample size and general population of elective surgery patients, this study is uniquely poised to compare the two methods of patient-reported change in quality of life and to determine the difference in quality of life score that surgical patients can detect. Determining the most accurate way to ascertain patient-reported quality of life can support elective surgery decisions and future studies of quality of life.\n\n\nSpecific aims\n\nAim 1\n\nThe primary aim of this study is to compare self-reported change in quality of life (better/same/worse) to the change in a validated (VR-12) quality of life score (both physical and mental component scores), 30 days after elective surgery.\n\nWe hypothesize that the median physical and mental quality of life scores will be significantly higher in patients reporting “better” quality of life compared to those reporting “same,” and significantly higher in those reporting “same” quality of life to those reporting “worse.” We also hypothesize that the overall agreement will be “substantial” (kappa=0.61 to 0.80), with the majority of error occurring in patients whose validated measure showed a decline in quality of life, but who reported “same” or “better” quality of life. For that reason, we expect the percent agreement between the self-reported and validated scales to be lowest in the group reporting “better” quality of life after surgery.\n\nAim 2\n\nA secondary aim is to compare the change in physical and mental quality of life scores that patients were able to perceive to the MCID established in literature.\n\nWe hypothesize that the difference in quality of life that our surgical patients can detect will be similar to the difference reported in the literature (ie a 5-point change).\n\nAim 3\n\nAnother secondary aim is to describe the change in physical and mental quality of life for both methods across surgical specialties.\n\nWe hypothesize that the change in quality of life will be greater for specialties correcting limited problems such as orthopedic and plastic surgery, while the change will be lower for specialties with more complex problems such as neurosurgery and cardiothoracic surgery.\n\n\nStudy design\n\nThis prospective, observational cohort study is a sub-study of the Systematic Assessment and Targeted Improvement of Services Following Yearlong Surgical Outcomes Surveys (SATISFY-SOS) study. SATISFY-SOS is an ongoing registry that has been enrolling patients at Barnes Jewish Hospital since July, 201218. All enrolled patients complete a survey of baseline health during their visit to the preoperative assessment clinic and then complete a follow-up survey approximately 30 days after surgery (see Supplementary Material for these two surveys). The intervention for this study is to compare self-reported quality of life (“How would you rate your quality of life now? (better/same/worse)”) to the quantitative change in their VR-12 quality of life scores between the baseline survey and 30-day follow-up survey. All patients answer both questions and therefore serve as their own controls.\n\n\nStudy group\n\nThe target population is all patients undergoing elective surgery at Barnes Jewish Hospital who attended the center for preoperative assessment and planning between January 15, 2014 and October 7, 2015. Inclusion criteria include age 18 or older, ability to read the English consent form (see Supplementary Material for the consent form), ability to consent, and plans to undergo elective surgery. Over 70% of patients undergoing elective surgery are assessed by the center for preoperative assessment and planning (CPAP) clinic before surgery. Reasons for no assessment include urgent surgery, geographical limitations, or surgeon preference. Approximately 65% of all eligible patients consent to participate in SATISFY-SOS. Reasons for non-participation include patient refusal (~70% of cases), lack of nurse time or training (~20%), or lack of English literacy (~10%). A study comparing participants to non-participants showed no major differences in characteristics19. Approximately 92% of consented patients complete the baseline survey, and 60% of those respond to the 30-day follow-up survey.\n\nA total of 9,097 “complete” records (with both baseline and 30-day surveys) are available in the proposed time window. For the purposes of this sub-study, only the first complete record for each will be included in the final dataset (approximately 94% of the available records). This practice ensures that each record is statistically independent from all the other records. In addition, records with surgery to 30-day response dates of less than 20 days or more than 120 days will be excluded.\n\n\nRecruitment\n\nNurses at the CPAP clinic assess patient eligibility, recruit patients to participate, and obtain written consent. No payment is provided. While most patients decide whether or not to participate at this time, a patient can decide to participate any time between his or her CPAP visit and his or her surgery day. For patients who need special assistance, such as those who are blind or cannot physically sign a form, a witness can be obtained. However, in practice this rarely occurs. No arrangements are made for non-English speakers, mentally ill, children, or those suffering from dementia since those are excluded groups. If patients agree to participate, the CPAP nurse asks them to complete the baseline survey at the time of consent. Approximately 30 days following surgery, they receive a similar follow-up survey. Both surveys were designed to take 10 to 15 minutes to complete. The SATISFY-SOS research team holds monthly update meetings with all CPAP nurses to inform them about study progress and to encourage optimal recruitment.\n\n\nData\n\nAll preoperative and postoperative quality of life data comes from the SATISFY-SOS surveys, which are administered to patients at the preoperative assessment visit and then approximately 30 days after surgery. To maximize the follow-up survey response rate, patients are emailed the survey (once), mailed hard copy surveys (two times), and phoned (up to five times). The twelve items comprising the Veterans RAND 12 (VR-12) are items 24 through 35 on the survey, while the self-reported global quality of life question is item 1. The VR-12 is made up of two components: a physical component score (PCS), and a mental component score (MCS). Both scores are continuous on a scale from 0 to 100 (where higher is greater quality of life), and they are calibrated so that a score of 50 represents the US population mean20,21. The 30-day follow-up questionnaires also ask patients to self-report their change in quality of life. The question asks, “How would you rate your quality of life now?” with answer choices including “Better than before your procedure,” “The same as before your procedure,” and “Worse than before your procedure.” Surgical specialty for the procedure is obtained from the electronic medical record. Using queries in MetaVision (iMDsoft, Needham, MA), the informaticist will provide the requested survey and medical record data to the investigators. He performs rigorous data validation on each queried variable.\n\nSATISFY-SOS databases are hosted on a firewall-secured network server managed by the Department of Anesthesiology. The server is securely housed behind two locked doors within the departmental office suite and maintained and managed by the departmental IT team. Only the project Informaticist, Data Manager, and Director(s) have full access to these databases, which are also password-protected and encrypted for additional protection. Hardcopies of the baseline surveys are collected daily from the CPAP clinic and securely stored behind two locked doors within the Department of Anesthesiology. Baseline completed paper surveys are scanned into a digital image format (compressed TIFF). The digital image files are indexed and stored on a research file server that is attached to a private network with no public access. Survey email, mail and call lists are generated at Washington University in a similar manner to mailing lists for billing services. For each patient and date of service, a unique ID is generated and never duplicated. This unique ID is a nonsensical only meaningful to the research team.\n\nBaseline surveys are processed by Solutions Data Systems. The digital image files are transmitted to Solutions Data Systems via secure file transfer protocol. When data entry has been confirmed, Solutions Data Systems deletes the digital image file from their servers. Press Ganey, a vendor specializing in patient survey distribution and collection, disseminates, collects, and processes 30-day and 1-year surveys. Paper surveys processed through automated scanning are all manually checked, and a manager listens to 10% of telephone surveys. All telephone surveys are recorded and available for future quality checks for performance improvement. Press Ganey stores the survey hardcopies for 90 days while the study team conducts spot-check quality assessments of the scanned data. The company then shreds the paper copies. Similarly, Press Ganey will hold copies of the electronic files and electronic recordings for 90 days, after which the electronic files are removed permanently from their system (and then only maintained by Washington University). During this 90-day period, the study team conducts additional quality assessments of the converted data.\n\n\nStatistical considerations\n\nWe base sample size considerations for this study on the primary outcome. The first component of the primary endpoint is comparing the change in VR-12 quality of life scores among the three self-reported change groups (better/same/worse). Using a minimum important difference of five points, two tails, alpha of 0.05, and 80% power, the required sample size is 77 patients per group, or 231 total patients among the three groups. The second component of the primary outcome is the agreement between the two quality of life measures, as reflected by the kappa statistic. Kappa does not have sample size requirements beyond lack of sparse cells. The third component of the primary outcome is comparing the percentage agreement across the three different self-reported change groups. Since no studies have performed this type of comparison previously, we pre-specified a 10% change as the minimum important change. Estimating 80% agreement, and using two tails, alpha=0.05, and 80% power, the required total sample size is 311 per group, or 933 total patients. Therefore, this study has adequate power for all of these endpoints.\n\nThe following statistical analyses will be performed, using alpha=0.05 and 95 percent confidence intervals, where appropriate. All analyses will be performed twice, once for VR-12 physical quality of life, and again for the VR-12 mental quality of life.\n\nAim 1:\n\nCompare change in VR-12 QOL scores for those answering better/same/worse (Kruskal-Wallis). If significant, will use Wilcoxon Rank-Sum tests to compare each of the three groups, using a Bonferroni correction of alpha=0.017.\n\nCalculate overall agreement between the self-reported and validated quality of life measures (using weighted kappa, which penalizes disagreements in proportion to their seriousness, see Table 1)22.\n\nCalculate the overall percent of patients whose self-reported and validated quality of life scores matched (descriptive), including stratification by self-reported global change better/same/worse (compared using chi-square). For the purpose of this study, “matching” consists of:\n\n◦ Change in VR-12 > 0 for those responding their quality of life was “better”\n\n◦ Change in VR-12 between +5 and -5 for those responding their quality of life was the “same”\n\n◦ Change in VR-12 < 0 for those responding their quality of life was “worse”\n\nAim 2:\n\nCalculate the quality of life where an equal proportion of patients reported better and same quality of life (MCID for improvement), or same and worse quality of life (MCID for deterioration). This is an anchor-based approach23.\n\nAim 3:\n\nDescribe the change in VR-12 score and percent of patients reporting better, same, and worse quality of life for each of the following surgical specialties: neurosurgery, orthopedic, plastic, ophthalmologic, general, cardiac, gynecologic, otolaryngology, gastrointestinal/hepatobiliary, urologic, and “other.”\n\nOnly those answering both the self-reported quality of life question and at least ten out of twelve VR-12 questions at baseline and 30 days will be included. We will describe the characteristics of those with and without missing data. Multiple imputation will be used to fill in missing measurements for those missing two or fewer items on the VR-12 questionnaire. Analysis will be performed by VLK and MRJ after this protocol is submitted online.\n\n\nLimitations\n\nAlthough the “validated” VR-12 measure is based on a standardized scale that has been rigorously tested and studied, it is also self-reported and probably also contains bias. Because the VR-12 produces physical and mental component scores (PCS and MCS), but the self-reported question asks about overall quality of life, the observed association between the self-reported quality of life and the individual component scores might be artificially lowered. Also, the order of the questions on the surveys might influence patients’ responses by priming them. Since the self-reported question occurs first, it may alter responses on the VR-12 items, which occur later in the same survey. Another limitation is that postoperative quality of life is only measured at 30 days. It is possible that the relationship between the two measures is different at different time points. In addition, the 30-day follow-up survey is often completed near 30 days, but the time of completion ranges from 20 to 120 days after surgery. Nevertheless, the time point for completing the two quality of life scales is the same in each individual patient, so the main results of this study should not be affected.\n\nThis study includes just one academic medical center, and its patient population and rules for preoperative assessment clinic attendance may differ from other hospitals. In addition, only 65% of eligible preoperative assessment clinic attendees enrolled in the study, which may introduce bias. However, our analyses indicate that participants do not differ in important ways from non-participants19. Furthermore, even if the sample was maximally biased, enrolling 65% of the actual target population means the results contain at least 80% accuracy24. Another factor that biases the sample is nonresponse to the follow-up surveys. Though we mitigate nonresponse through an extensive follow-up protocol, only 60% of patients respond to the 30-day survey. Fortunately, our previous work shows that the characteristics of responders do not differ significantly from the characteristics of non-responders (unpublished manuscript). Finally, anchor-based approaches for calculating MCID can be influenced by recall bias and have been shown to more strongly reflect patients’ current health status than the true change over time23.\n\n\nCompliance\n\nSince the exposure for this study is patient-reported quality of life, no procedures for monitoring exposure compliance are necessary. Subjects are withdrawn from SATISFY-SOS if requested. The informaticist and Press Ganey are notified to ensure that the patient is no longer approached for data collection. As described in the consent form, data already collected may continue to be used.\n\n\nEthical considerations\n\nThis study is approved by the Institutional Review Board (Washington University Human Research Protection Office, IRB ID# 201505035). As a sub-study of SATISFY-SOS, it has a waiver of informed consent. Written, informed consent is obtained from all participants for SATISFY-SOS (IRB ID# 201203088). Since this study is survey-based, it involves no more than minimal risk to patients. As described above, no special allowances are made for non-English speakers, children, or mentally ill. Participants may withdraw from the study at any time.\n\n\nFinance and insurance\n\nFinance details, insurance details, and cover for negligent and non-negligent harm are not relevant for this study since it involves no more than minimal risk to patients. Patients receive no compensation for participation.\n\n\nReporting and dissemination\n\nResults of this study will be presented at national meetings and published in a scientific journal. Participants will be individually notified of results only if discoveries are made that directly impact their health. The data and code for this project will be available upon email request.",
"appendix": "Author contributions\n\n\n\nVLK conceived the study protocol. MRJ provided statistical expertise. MSA provided SATISFY-SOS expertise. 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\nVLK was supported by the Washington University Institute of Clinical and Translational Sciences grant UL1TR000448 from the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health (NIH). MSA was supported by grant 1UH2AG050312-01 from the National Institute on Aging, grant BJHF#7937-77 from the Barnes-Jewish Hospital Foundation, and the Washington University Department of Anesthesiology. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary Material\n\nBaseline survey.\n\nQuestionnaire provided to patients during their preoperative assessment visit\n\nClick here to access the data.\n\n30-day survey.\n\nFollow-up questionnaire sent to patients approximately 30 days after surgery\n\nClick here to access the data.\n\nConsent form.\n\nConsent form for SATISFY-SOS.\n\nClick here to access the data.\n\n\nReferences\n\nGuidelines for completing a research protocol for observational studies. Biostatistics Group, UCLH/UCL/RFH Biomedical Research Unit; 2010. Reference Source\n\nClancy C, Collins FS: Patient-Centered Outcomes Research Institute: the intersection of science and health care. Sci Transl Med. 2010; 2(37): 37cm18. PubMed Abstract | Publisher Full Text\n\nWaljee J, McGlinn EP, Sears ED, et al.: Patient expectations and patient-reported outcomes in surgery: a systematic review. Surgery. 2014; 155(5): 799–808. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuk KS, Baek JH, Park JO, et al.: Postoperative quality of life in patients with progressive neuromuscular scoliosis and their parents. Spine J. 2015; 15(3): 446–453. PubMed Abstract | Publisher Full Text\n\nTan TL, Le Duff MJ, Takamura KM, et al.: Do clinical and quality of life scores change over time after hip resurfacing? Hip Int. 2015; 25(2): 146–151. PubMed Abstract | Publisher Full Text\n\nSkirko JR, Weaver EM, Perkins JA, et al.: Change in Quality of Life with Velopharyngeal Insufficiency Surgery. Otolaryngol Head Neck Surg. 2015; 153(5): 857–864. PubMed Abstract | Publisher Full Text\n\nBrown LK, Waljee AK, Higgins PD, et al.: Proximity to disease and perception of utility: physicians' vs patients' assessment of treatment options for ulcerative colitis. Dis Colon Rectum. 2011; 54(12): 1529–1536. PubMed Abstract | Publisher Full Text\n\nBoyd NF, Sutherland HJ, Heasman KZ, et al.: Whose utilities for decision analysis? Med Decis Making. 1990; 10(1): 58–67. PubMed Abstract | Publisher Full Text\n\nUbel PA, Loewenstein G, Jepson C: Disability and sunshine: can hedonic predictions be improved by drawing attention to focusing illusions or emotional adaptation? J Exp Psychol Appl. 2005; 11(2): 111–123. PubMed Abstract | Publisher Full Text\n\nSmith D, Loewenstein G, Jepson C, et al.: Mispredicting and misremembering: patients with renal failure overestimate improvements in quality of life after a kidney transplant. Health Psychol. 2008; 27(5): 653–658. PubMed Abstract | Publisher Full Text\n\nMather M, Shafir E, Johnson MK: Misremembrance of options past: source monitoring and choice. Psychol Sci. 2000; 11(2): 132–138. PubMed Abstract | Publisher Full Text\n\nUbel PA, Loewenstein G, Schwarz N, et al.: Misimagining the unimaginable: the disability paradox and health care decision making. Health Psychol. 2005; 24(4 Suppl): S57–62. PubMed Abstract | Publisher Full Text\n\nSmith DM, Sherriff RL, Damschroder L, et al.: Misremembering colostomies? Former patients give lower utility ratings than do current patients. Health Psychol. 2006; 25(6): 688–695. PubMed Abstract | Publisher Full Text\n\nNorman GR, Sloan JA, Wyrwich KW: Interpretation of changes in health-related quality of life: the remarkable universality of half a standard deviation. Med Care. 2003; 41(5): 582–592. PubMed Abstract | Publisher Full Text\n\nFarivar SS, Liu H, Hays RD: Half standard deviation estimate of the minimally important difference in HRQOL scores? Expert Rev Pharmacoecon Outcomes Res. 2004; 4(5): 515–523. PubMed Abstract | Publisher Full Text\n\nZhou F, Zhang Y, Sun Y, et al.: Assessment of the minimum clinically important difference in neurological function and quality of life after surgery in cervical spondylotic myelopathy patients: a prospective cohort study. Eur Spine J. 2015; 24(12): 2918–2923. PubMed Abstract | Publisher Full Text\n\nParker SL, Adogwa O, Mendenhall SK, et al.: Determination of minimum clinically important difference (MCID) in pain, disability, and quality of life after revision fusion for symptomatic pseudoarthrosis. Spine J. 2012; 12(12): 1122–1128. PubMed Abstract | Publisher Full Text\n\nAvidan MS: Systematic Assessment and Targeted Improvement of Services Following Yearlong Surgical Outcomes Surveys (SATISFY-SOS). In: ClinicalTrials.gov: [cited 2016 Feb 10]. Reference Source\n\nHelsten DL, Abdallah AB, Avidan MS, et al.: Methodological considerations for collecting patient reported outcomes from unselected surgical patients. Anesthesiology. 2016; In Press.\n\nKazis LE, Miller DR, Skinner KM, et al.: Applications of methodologies of the Veterans Health Study in the VA healthcare system: conclusions and summary. J Ambul Care Manage. 2006; 29(2): 182–188. PubMed Abstract | Publisher Full Text\n\nSelim AJ, Rogers W, Fleishman JA, et al.: Updated U.S. population standard for the Veterans RAND 12-item Health Survey (VR-12). Qual Life Res. 2009; 18(1): 43–52. PubMed Abstract | Publisher Full Text\n\nSim J, Wright CC: The kappa statistic in reliability studies: use, interpretation, and sample size requirements. Phys Ther. 2005; 85(3): 257–268. PubMed Abstract\n\nWright A, Hannon J, Hegedus EJ, et al.: Clinimetrics corner: a closer look at the minimal clinically important difference (MCID). J Man Manip Ther. 2012; 20(3): 160–166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStamatopoulos C: Observations on the geometric properties of accuracy growth in sampling with finite populations. Rome: Food and Agriculture Organization of the United Nations; 1999. Reference Source"
}
|
[
{
"id": "15801",
"date": "05 Sep 2016",
"name": "Anna Woodbury",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 primary objective of this study is to perform a sub-study of the Systematic Assessment and Targeted Improvement of Services Following Yearlong Surgical Outcomes Surveys (SATISFY-SOS) study by comparing a validated quality of life measure (VR-12) to a self-reported \"global\" quality of life question.\n\nSecondary aims include comparing mental and physical quality of life pre- and post- surgery (Aim 2) and to describe these based on types of surgeries (Aim 3). The authors feel this is important because self-reported measures are subject to recall bias, and yet are more commonly used.\nMajor criticisms: None\nMinor criticisms:\nCo-morbidities (such as depression, Alzheimer's, or other psychiatric or neurologic illness) and their treatment or lack of treatment may affect quality of life scores. Will these be accounted for and will the patient be monitored? For example, if a patient suffers from depression, undergoes surgery, and is started on an antidepressant after surgery, this would potentially affect quality of life, but is not directly related to the surgery itself. I would like to know how this type of confounding variable will be addressed.\n\nThe validated tool used should be compared also with the PROMIS measures, which are essentially individually validated questions, often reliant on patient self-report. Would consider adding these.",
"responses": []
},
{
"id": "15800",
"date": "08 Sep 2016",
"name": "Meghan Lane-Fall",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this report, Kronzer and colleagues detail a study protocol for an observational study of patient quality of life after elective surgery. They intend to use two methods to assess quality of life: (1) a validated scale and (2) a simple closed-ended question about whether their quality of life is better, worse, or the same as that before surgery. In the interest of research transparency, I applaud the authors on publishing their study protocol in advance of proceeding with their work. This will allow reviewers and readers of subsequent reports to determine whether the investigators adhered to their study protocol.\nManuscript comments: The manuscript is clearly written and easy to understand. The data collection approach and analytic procedures are reasonable, and the authors have adequately addressed potential threats to validity (e.g. non-response bias). It is unfortunate that limited English speakers are excluded from the study population, but this is a limitation of the registry overall, not just this sub-study.\nMajor criticisms:\nThe authors state in their Limitations section that some of the surveys are completed as late as 120 days after surgery (instead of 30 days after). I worry that the relationship between the VR-12 score and the single global QOL question may not be consistent across time, which could bias the results. Have the authors considered restricting their sample to patients who complete the follow-up survey within a certain range of time (e.g. 20-40 days)?\nMinor criticisms:\nI would define VR-12 earlier in the manuscript. It is currently in the Data section, but should be moved up to its first mention in the Methods section.\n\nIn the Data section, the authors state that the informaticist \"performs rigorous data validation on each queried variable\", but do not specify what the data validation procedures are. Some limited additional detail here would be useful.\n\nInstrument comments:\nMinor criticism:\nIn the baseline instrument, both \"circle all\" (Question 1) and \"check all\" (Question 2) are used - these should be the same for consistency.\nReferences:\nIn Reference 19, the title is incorrect. \"Methodological\" should be \"Methodologic\", and the citation should be updated with publication information: Anesthesiology. 2016 Sep;125(3):495-504. doi: 10.1097/ALN.0000000000001217.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-976
|
https://f1000research.com/articles/5-962/v1
|
23 May 16
|
{
"type": "Software Tool Article",
"title": "IncucyteDRC: An R package for the dose response analysis of live cell imaging data",
"authors": [
"Philip J. Chapman",
"Dominic I. James",
"Amanda J. Watson",
"Gemma V. Hopkins",
"Ian D. Waddell",
"Donald J. Ogilvie",
"Dominic I. James",
"Amanda J. Watson",
"Gemma V. Hopkins",
"Ian D. Waddell",
"Donald J. Ogilvie"
],
"abstract": "We present IncucyteDRC, an R package for the analysis of data from live cell imaging cell proliferation experiments carried out on the Essen Biosciences IncuCyte ZOOM instrument. The package provides a simple workflow for summarising data into a form that can be used to calculate dose response curves and EC50 values for small molecule inhibitors. Data from different cell lines, or cell lines grown under different conditions, can be normalised as to their doubling time. A simple graphical web interface, implemented using shiny, is provided for the benefit of non-R users. The software is potentially useful to any research group studying the impact of small molecule inhibitors on cell proliferation using the IncuCyte ZOOM.",
"keywords": [
"R package",
"Drug discovery",
"Dose response curve",
"Live cell imaging",
"Oncology",
"Shiny"
],
"content": "Introduction\n\nLive cell imaging permits cell proliferation to be monitored in real time over a period of days or weeks, and the IncuCyte ZOOM from Essen Biosciences (http://www.essenbioscience.com/essen-products/incucyte/) is a platform used for such experiments. In oncology drug discovery the proliferation of cancer cell lines can be monitored in the presence of different small molecule inhibitors at different concentrations to gain insight as to the efficacy and mechanism of action of novel compounds. This approach provides a useful alternative to measuring cell proliferation as a phenotypic endpoint after a fixed period such as 3 days. It has the advantage of being able to visualise graphically and morphologically when treatments impact on proliferative capacity. Furthermore, vehicle dosed cells can be used to control for population doubling resulting in normalisation across many different cell lines. This may be particularly important when downstream effects of compound treatment require a round of replication before they are apparent.\n\nThe IncuCyte ZOOM comes with sophisticated software (http://www.essenbioscience.com/essen-products/software/incucyte-base-software/) for the analysis of imaging data and the calculation of metrics such as percent confluence, however we sought a more streamlined workflow for the analysis of data from cell-based compound screening assays. The result was the development of the R1 package IncucyteDRC which permits quick and easy fitting of dose response curves and calculation of EC50 values, as well as the export of data to standard analysis packages such as GraphPad PRISM http://www.graphpad.com/scientific-software/prism/ and Dotmatics Studies http://www.dotmatics.com/products/studies/. In addition, a graphical user interface was developed as part of the package using Shiny2, as well as a novel algorithm for normalizing data by cell doubling time.\n\n\nMethods\n\nA basic workflow for the IncucyteDRC package is outlined below:\n\n1. Import data and plate map information\n\n2. Create an IncucyteDRCSet object\n\n3. Fit growth curves to the data\n\n4. Calculate the growth curve values at a given ‘cut time’\n\n5. Carry out a dose response analysis using the extracted values\n\nOptionally, the cut time can be calculated automatically for a required number of doublings.\n\nThe importPlatemapXML function imports .Platemap files generated by the IncuCyte ZOOM software to define the contents of a plate. The function can extract the following parameters: compound (description, concentration and units), growth condition (description), and cell type (description, passage and seeding density). These are converted into a data.frame object which is used as a basis for further analysis. Alternatively, the importPlatemap function can import a data.frame or a tab delimited text file but these must be in the correct format - see example(importPlatemap).\n\nThe importIncucyteData function imports data generated by the Incucyte Zoom software, as described in detail in the package vignette entitled ’Exporting From Incucyte Zoom Software’. The output is a IncucyteDRCPlateData S3 object.\n\nThe IncucyteDRCSet object is at the centre of the package workflow. Subsequent functions add to and operate on the data contained within the object. It can be initiated using the makeIncucyteDRCSet function and at its simplest combines a plate map data frame generated by importPlatemapXML function with an IncucyteDRCPlateData object created by the importIncucyteData function. In addition, a cut time can be specified, and a single row metadata data.frame provided.\n\nA key attribute of an IncucyteDRCSet is that all data share a common control growth curve. Thus it is appropriate to have an IncucyteDRCSet object containing multiple compounds on a common cell line background, but if the cell line background changes then one object is needed per condition.\n\nThe splitIncucyteDRCPlateData function is a convenience function that splits up an IncucyteDRCPlateData object into a list of IncucyteDRCSet objects if necessary, or a single IncucyteDRCSet object if not. It also automatically populates the metadata element. Therefore it is sensible to use this function, rather than the makeIncucyteDRCSet function, in a standard workflow.\n\nThe first step in the analysis process is to fit growth curves to the data using the base R loess function. This is done using the related functions fitGrowthCurvesGrouped and fitGrowthCurvesIndividual. The former combines replicates to create a single growth curve for a given sample and compound concentration, whilst the latter fits a growth curve for every single well on a plate. The IncucyteDRCSet object is updated with four dplyr3 data frames (tbl_df objects) containing the models themselves and the fitted data for plotting. Refer to the dplyr3 package documentation for more information on how models and other objects can be stored in data frames.\n\nOnce the growth curves have been fitted, they can be visualised using the plotIncucyteDRCSet function (Figure 1).\n\nThe cut time is represented as a blue dashed vertical line.\n\nAlthough the growth curves themselves can give some insight into the effect that different compounds have on the growth of the cell line, to analyse this formally the growth parameter can be determined at a specific ‘cut time’ using the calculateDRCData function. This data is stored in the IncucyteDRCSet object and can subsequently be exported to various formats using the exportDRCDataToDataFrame, exportDRCDataToPRISM and exportDRCDataToDotmatics functions. These data can then form the basis of a dose response analysis to generate an EC50 value.\n\nIt is advisable to export the dose response data and fit the curves in specialist software such as GraphPad PRISM or Dotmatics Studies, but for convenience there are also functions that utilise the drc package4 to provide an end-to-end workflow. The fitDoseResponseCurve function generates models for each compound in the IncucyteDRCSet (using the drc::LL.4 function) and calculateEC50 predicts an EC50 value for each model (using the drc::EC50 function). The exportEC50Data function generates a data.frame containing the values stored in the IncucyteDRCSet object, whilst the dose response curves can be visualised with the plotDoseResponseCurve function.\n\nThe y-axis is the growth value (percent confluence in this case) whilst the x-axis is the micromolar compound concentration\n\nIn the previous examples, a cut time has been manually provided following an inspection of the growth curves. Typically the cut time should be as long as possible to allow differences to become pronounced, but still during the exponential phase of cell growth before the cells become confluent. However, sometimes it might be desirable to determine the cut time for a given number of doublings, so that the EC50 for fast and slow growing cell lines can be normalised.\n\nThe calculateCutTimeForIDRCSet function can be run on an IncucyteDRCSet object to determine the optimal cut time from the control (vehicle treated) growth curves given a set of parameters. These include baseline_time (the timepoint to take as a baseline for calculating doublings), no_doublings (the number of doublings required) and max_val (the maximum allowable growth curve value). The algorithm works out the growth curve value at the baseline time and what value would correspond to a given number of doublings from this point, then returns the timepoint at which this value is achieved. If this value exceeds the maximum value, then the time at which the maximum value is returned instead, along with the actual number of doublings achieved.\n\nIn detail, the growth curve is truncated by removing its non-exponential phase along with any part exceeding the user defined maximum (usually 80%). The non-exponential phase is defined as the part of the curve after the timepoint at which the n=30 moving average of the second differences of the growth values reaches its minimum. This corresponds to the time at which the rate of cell growth is slowing most rapidly. The growth value required to reach the desired number of doublings from a user defined baseline time was calculated (using the predict function on the loess model object), and the elapsed time to reach this is returned as the cut time. If the required growth value is not reached on the truncated growth curve, then the maximum growth value achieved on the truncated curve is returned instead along with the actual number of doublings that occurred.\n\nThe black dashed lines represent user-defined parameters whilst the blue dashed lines are calculated by the algorithm. The blue vertical dashed line is the cut time.\n\nThe IncucyteDRC R package is available on CRAN and is built on top of R version 3.2.31 and Shiny 0.13.22 and has no special R dependencies beyond packages that are available on CRAN and so should work on any operating system supported by R. A full list of R package dependencies is present in the Description file of the package.\n\nThe package can be installed from CRAN:\n\ninstall.packages(’IncucyteDRC’)\n\nThe latest development version can be installed in an R session from GitHub using the devtools5 package:\n\ndevtools::install_github(’chapmandu2/IncucyteDRC’)\n\nHaving successfully installed the IncucyteDRC package, it can be loaded using library(’IncucyteDRC’) and a detailed vignette is available which can be viewed by typing browseVignettes(’IncucyteDRC’). Help and examples are available for functions within the package e.g.\n\n?importIncucyteData\n\nexample(importIncucyteData)\n\nFinally, a Shiny app2 provides a graphical user interface for the functionality within the package. This can be launched from within R using shinyVisApp() whilst details for how to set up the IncucyteDRC package as a Shiny app on a Shiny server is included in the vignette.\n\n\nUse cases\n\nThe package vignette contains detailed use cases using two example datasets. These can be loaded by running the examples for the importPlatemapXML and importIncucyteData functions:\n\nexample(’importPlatemapXML’)\n\nexample(’importIncucyteData’)\n\n\nSummary\n\nThe IncucyteDRC R package provides a simple, reproducible workflow for the dose response analysis of live cell imaging data from the IncuCyte Zoom instrument. To facilitate its use by non-R users, a comprehensive and user-friendly graphical interface is provided as a Shiny2 web application. The software is freely available on CRAN and is potentially useful to any research group studying the impact of small molecule inhibitors on cell proliferation.\n\n\nSoftware availability\n\n1. Software available from: https://cran.r-project.org/web/packages/IncucyteDRC/\n\n2. Latest source code: https://github.com/chapmandu2/IncucyteDRC/\n\n3. Archived source code as at time of publication: http://dx.doi.org/10.5281/zenodo.512606\n\n4. License: GPL-2",
"appendix": "Author contributions\n\n\n\nPJC conceived and wrote the software and wrote the manuscript. DJ, MW, GH designed and executed experiments, analysed data, and provided feedback on functionality. DJ, IW, DO conceived the cell doubling normalisation concept. All authors helped review the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by Cancer Research UK (Grant numbers C480/A1141 and C5759/A17098, Principal Investigator Donald Ogilvie).\n\n\nAcknowledgments\n\nThe authors would like to thank Chris Campbell of Mango Solutions for technical assistance in preparing the R package for its submission to CRAN.\n\n\nReferences\n\nR Core Team: R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, 2015. Reference Source\n\nChang W, Cheng J, Allaire JJ, et al.: shiny: Web Application Framework for R. 2016; R package version 0.13.2. Reference Source\n\nWickham H, Francois R: dplyr: A Grammar of Data Manipulation. 2015; R package version 0.4.3. Reference Source\n\nRitz C, Streibig JC: Bioassay analysis using R. J Stat Softw. 2005; 12(5). Publisher Full Text\n\nWickham H, Chang W: devtools: Tools to Make Developing R Packages Easier. 2015; R package version 1.9.1. Reference Source\n\nChapman P: IncucyteDRC: Version 0.5.4: F1000R and CRAN release. Zenodo. 2016. Data Source"
}
|
[
{
"id": "15204",
"date": "03 Aug 2016",
"name": "Guillaume Devailly",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nP. Chapman et al. developed an R package to analyse data obtained from the Incucyte instrument, and specifically growth curve responses to various treatment. While one could interpret Incucyte data without this package, or without any programming skills at all, there is no doubt that Incucyte users knowing R, or intensive Incucyte users wishing to automatize their analysis, will greatly benefit from using it. Overall the package is working well, is easily available through the popular CRAN repository, is well documented, with notably nice vignette, a video tutorial and a companion shiny app. The present manuscript present this package appropriately.\nI have no major concern with it, and only small comments and suggestions for improvement. About the manuscript:\nPackage availability on CRAN could be mentioned in the abstract. Authors could detail the reasons why they advise against using their package for carrying dose response analysis, in favour of alternative software. Page 2: “A key attribute of an IncucyteDRCSet is that all data share a common control growth curve.” I was a bit confused at first as I interpreted this as the non-support of replicates for the negative control. It could be mentioned that the control growth curve can (and should) come from multiple replicated control wells. Since the public shiny app is now available, it could be mentioned in the manuscript, and even in the abstract, if the authors are willing to support it for a few years.\nAbout the package (I tested only the CRAN version IncucyteDRC_0.5.4, what follows might not apply to future version, or to even to the github version.):\nThe importPlatemap and importPlatemapXML functions has a default value “DMSO” as a control_cpd parameter. I feel this is a terrible idea as users might at first not be aware of this crucial parameter. Not everyone use DMSO as a control, and some silly users might even have named it “dmso” in their incucyte data. Notably it is not used (left as default) in the first usage of the importPlatemapXML function in the “Overview of the IncucyteDRC Package” vignette. I would advise to not use any default value for this parameter and throw an error if it is missing. The importIncucyteData helps mentioned that you wanted to add little more details about it, you might want to have a look (“NEED MORE DOCUMENTATION HERE ABOUT HOW TO DO EXPORT!!”). The help pages for functions might benefit form a “suggest/see also” section (i.e. linking back the various import and export functions). The IncucyteDRCSetList object is quite useless at the moment. Could you overload the fitGrowthCurvesGrouped, fitGrowthCurvesIndividual, calculateCutTimeForIDRCSet, calculateDRCData, fitDoseResponseCurve, and calculateEC50 function so that when provided with an IncucyteDRCSetList object, they return an IncucyteDRCSetList object in which each IncucyteDRCSet object has been modified? That would be a bit more convenient for the user than the apply method you suggest in the vignette. I got an error with the local version of the shiny app when testing it with the example 2 files (Error in match.arg: 'arg' must be NULL or a character vector). Since it does not affect the shinyapps.io version, I guess it might be already fixed in github, or it might be a local issue.",
"responses": []
},
{
"id": "16099",
"date": "16 Sep 2016",
"name": "Clara Alsinet-Armengol",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have developed an R package to visualise drug response data and make some basic calculations from experiments performed on the Incucyte ZOOM system. The funtions are easy to use and, interestingly, the useful IncucyteDRCSet object has been designed so that it can progressively integrate all data generated. Both the article and the R documentation are clearly explained.\nI would just make one suggestion on the manuscript. One of the features of the software is that it allows for normalization of the drug response by cell doubling time. A brief description of its relevance could be added.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-962
|
https://f1000research.com/articles/5-951/v1
|
20 May 16
|
{
"type": "Review",
"title": "Toxic epidermal necrolysis",
"authors": [
"Wolfram Hoetzenecker",
"Tarun Mehra",
"Ieva Saulite",
"Martin Glatz",
"Peter Schmid-Grendelmeier",
"Emmanuella Guenova",
"Antonio Cozzio",
"Lars E. French",
"Tarun Mehra",
"Ieva Saulite",
"Martin Glatz",
"Peter Schmid-Grendelmeier",
"Emmanuella Guenova",
"Antonio Cozzio"
],
"abstract": "Toxic epidermal necrolysis (TEN) is a rare, life-threatening drug-induced skin disease with a mortality rate of approximately 30%. The clinical hallmark of TEN is a marked skin detachment caused by extensive keratinocyte cell death associated with mucosal involvement. The exact pathogenic mechanism of TEN is still uncertain. Recent advances in this field have led to the identification of several factors that might contribute to the induction of excessive apoptosis of keratinocytes. In addition, specific human leukocyte antigen types seem to be associated with certain drugs and the development of TEN. As well-controlled studies are lacking, patients are treated with various immunomodulators (e.g. intravenous immunoglobulin) in addition to the best supportive care.",
"keywords": [
"Toxic epidermal necrolysis",
"skin detachment",
"keratinocyte cell death",
"Stevens-Johnson syndrome"
],
"content": "Introduction\n\nThe exposure to drugs has increased with demographic shifts associated with a higher morbidity of the population. Along with this phenomenon, a rise in the incidence of adverse drug reactions (ADRs) has been observed. Toxic epidermal necrolysis (TEN) is a rare, acute, and life-threatening mucocutaneous disease that is usually drug related. Recent evidence situates TEN as the most severe form amongst a spectrum of severe epidermolytic adverse cutaneous drug reactions, which further include Stevens-Johnson syndrome (SJS) and the SJS-TEN overlap disease1. TEN is a consequence of extensive keratinocyte cell death that results in the separation of significant areas of skin at the dermal-epidermal junction with the production of bullae followed by skin sloughing. This extensive cell death also leads to mucous membrane detachment and contributes to the characteristic symptoms of TEN, which include high fever, mucositis, and moderate to severe skin pain, anxiety, and asthenia. Although the pathogenic mechanism of TEN remains incompletely understood, significant progress in this field of medicine has been made in recent years. The improvements range from the clinical classification that is essential for a better understanding of this disorder to the identification of genetic susceptibilities to certain drugs and the implementation of the first preventive genetic screening measures for selected patient groups and drug classes1. This review aims to provide an up-to-date overview of TEN, emphasizing pathogenesis and immunopathology.\n\n\nHistory and epidemiology\n\nThe first description of TEN was made by the Scottish dermatologist Alan Lyell in 19562. This severe skin disease, also referred to as Lyell’s syndrome, was initially considered to be a toxic eruption, which closely resembles a severe burn or scalding of the skin2. The skin lesion-associated erythematous plaques and widespread areas of epidermal detachment were referred to by Dr. Lyell as necrolysis. He also described an involvement of the mucous membranes as part of the syndrome and noted that there was very little inflammation in the dermis, a feature that was later referred to as “dermal silence”3. TEN was only associated2 with exposure to certain medications as more patients presenting with TEN were reported subsequent to Lyell’s original publication.\n\nTEN is a rare disease with an annual incidence of approximately 0.4–1.2 cases per million individuals4,5. There are several factors that seem to impact the incidence of SJS and TEN; regional differences in drug prescription patterns, the population’s genetic background such as human leukocyte antigen (HLA) status and phenotypes of metabolizing enzymes, co-occurrence of cancer, frequency of radiotherapy, and prevalence of certain infectious diseases such as HIV are associated with an increased incidence of TEN6,7.\n\n\nClinical features\n\nThe main symptoms of TEN are usually preceded by non-specific symptoms such as fever, stinging eyes, and discomfort upon swallowing by several hours up to several days. Characteristically, cutaneous lesions first appear in the presternal region as well as the face, palms, and soles of the feet. Mucosal involvement occurs in more than 90% of patients, predominantly affecting the mouth, genitalia, and/or ocular region. In some cases, the respiratory system and gastrointestinal tract are also affected. The morphology of lesions is characterized by erythema and erosions8,9. Ocular involvement is frequent10,11. Early cutaneous lesions frequently present as livid, erythematous maculae: they may or may not show signs of slight infiltration. During the course of the disease, the lesions rapidly coalesce and become tense bullae (Figure 1). With disease progressions, they form large confluent areas of epidermal detachment. The degree of skin involvement is a highly important prognostic factor. Skin involvement should be determined including only already detached necrotic (e.g. blisters or erosions) or detachable skin (Nikolsky positive). A classification system for SJS and TEN according to the extent of skin detachment has been suggested by Bastuji Garin et al.12:\n\n1–10%: SJS\n\n11–30%: SJS-TEN overlap disease\n\n>30%: TEN\n\n(A) Skin detachment with facial erosions, including involvement of the lips and conjunctiva. (B) TEN with an extensive cutaneous involvement marked by detached and detachable apoptotic skin erosions on the trunk.\n\nFurthermore, to predict the risk of death in TEN patients, the TEN-specific severity of illness score (SCORTEN) has been proposed13.\n\nSJS and TEN frequently leave cutaneous sequelae after healing. These include cutaneous hyperpigmentation and hypopigmentation (62.5% of cases), nail dystrophy (37.5% of cases), and ocular complications (50% of cases)14,15.\n\nIn most cases of TEN, a strong, direct association of the disease with preceding drug consumption can be established. Indeed, preceding exposure to medications is reported in over 95% of patients with TEN, and a strong association between drug ingestion and cutaneous manifestation is observed in 80% of cases1. Approximately 100 compounds have been identified as the likely triggers of TEN so far, the most frequent being allopurinol, antibiotics, nonsteroidal anti-inflammatory drugs, and anticonvulsants1,16.\n\n\nPathogenesis\n\nSo far, the precise molecular and cellular pathogenic mechanisms leading to the development of SJS/TEN can be only partially explained. It is thought to be initiated by an immune response to an antigenic drug-host tissue complex9,17–20. Current scientific opinion proposes three different hypotheses as to the formation of the antigenic complex (Figure 2): i) covalent binding of the drug to a peptide of the cellular surface (hapten/pro-hapten concept); ii) non-covalent, direct interaction of the drug with a specific major histocompatibility complex (MHC) class I allotype (p-i concept); and iii) presentation of an altered-self repertoire by direct drug-MHC I interaction (altered peptide concept). The first, the well-known hapten model, is far less likely to be HLA restricted. However, the two remaining concepts do favor specific HLA phenotypes. According to the latter two hypotheses, a pharmacological agent serving as the allergen would directly bind to specific HLA molecules and/or T cell receptors without prior processing by antigen-presenting cells. In the case of the p-i concept, the mere pharmacological interaction of certain drugs with immune receptors would be sufficient to induce a drug hypersensitivity reaction21–24. Additionally, recent publications have shown that the HLA-peptide repertoire can be modified by abacavir and carbamazepine, resulting in enhanced peptide presentation and increased autoimmune reactivity (altered peptide model)24,25. Besides, it has been suggested that SJS may be induced via direct interaction between carbamazepine and HLA-B*150226,27. However, the identification of specific drug-related HLA alleles that strongly increase the likelihood of developing SJS or TEN strengthens the hypothesis of the genetic susceptibility of patients to TEN, supporting the concept of HLA-restricted drug presentation28–30. This finding is clinically relevant, as screening for the HLA-B*1502 allele in Asian patients prior to drug intake may identify persons at risk of developing severe epidermolytic adverse cutaneous drug reactions, for example in the case of carbamazepine-induced SJS or TEN31. Evidence suggests that immune activation by the drug-host tissue complex induces a strong expression of Fas-L, a cytolytic molecule, on keratinocytes as well as granulysin and annexin A1 secretion by CTLs, NK cells, NKT cells, and monocytes32–37 (Figure 3). As a result, Fas-L- and granulysin-mediated apoptosis and/or annexin-dependent necroptosis of keratinocytes with subsequent epidermal necrosis and detachment develop. This indicates that the disturbance of the balance between pro-inflammatory and immunomodulatory mechanisms may critically determine the clinical outcome in cutaneous inflammation. Interestingly, Th17 cells were found alongside CD8+ T cells in the blister fluid of SJS/TEN patients, but not in patients with erythema multiforme major (EMM). CD8+ T cells are a source of IL-17, which is a cytokine that promotes the recruitment of neutrophils38. Involvement of skin homing Th17 cells in SJS/TEN is suggested by an observed decrease in the periphery upon treatment-related disease improvement. Recent findings suggest that Th17 cells may alter their phenotype and become regulatory T cells39. Furthermore, recently it has been proposed that Th17 cells originally infiltrate skin lesions in SJS/TEN with regard to the described presence of granulysin-expressing drug-reactive Th17 cells40. The decrease in Th17 cells in patients with resolving SJS/TEN could therefore be associated with a simultaneous rise in regulatory T cells. This hypothesis should be examined in future studies. Neutropenia is generally associated with a higher mortality in SJS/TEN patients41.\n\n(A) Drugs inducing an adverse skin reaction are not antigenic by themselves. Instead, their immunogenicity may result from binding to carrier proteins, which allows the formation of neoantigens that are recognized by T cells upon presentation by antigen-presenting cells (APCs). (B) The p-i concept is based on the pharmacological interactions of drugs with immune receptors. Consistent with this concept, chemically inert drugs, which are unable to bind covalently to proteins, may activate specific T cells by binding directly to T cell receptors and/or major histocompatibility complex molecules. (C) The association of peptides with HLA molecules is highly specific. According to the “altered peptide model”, specific HLA molecules form a complex with certain drugs, thereby modifying the pool of self-peptides presented to T cells. This may result in increased autoimmunity. Concepts for immunological responses of SJS/TEN modified from Abe et al.52.\n\n(A) The causative medication might induce upregulation of Fas-L by keratinocytes constitutively expressing Fas, leading to activation of a death receptor-mediated apoptotic pathway. (B) The drug might interact with major histocompatibility complex class I-expressing cells, causing drug-specific CD8+ cytotoxic T cells to accumulate within epidermal blisters, releasing perforin and granzyme B that can kill keratinocytes. (C) Drug-activated monocytes secrete annexin A1, which induces necroptosis in keratinocytes. (D) The drug may also trigger the activation of CD8+ T cells, NK cells and NKT cells to secrete granulysin, which can induce keratinocyte death without the need for cell contact. This figure has been modified from French et al.1. (APC, antigen-presenting cell; NK cell, natural killer cell; NKT cell, natural killer T cell).\n\n\nTreatment\n\nRapid histological examination including direct immunofluorescence analysis of a lesional skin biopsy is paramount in the diagnostic work-up of SJS/TEN, as it helps to rule out diagnoses that can imitate TEN clinically. Autoimmune blistering diseases, fixed drug eruption (FDE), acute generalized exanthematous pustulosis (AGEP), and staphylococcal scalded skin syndrome (SSSS) should be ruled out (Table 1). The suspect drug should be discontinued immediately and supportive therapy should be ensured in the burn or intensive care unit42. However, valid data on effective therapeutic options are poor, and prospective controlled clinical trials, which can clearly show the benefit of certain treatment options, are lacking. Some case reports and case series report a benefit of cyclosporine, cyclophosphamide, N-acetylcysteine, TNF-α antagonists (e.g. etanercept and infliximab), systemic corticosteroids (favoring pulsed corticosteroid treatment), thalidomide, plasmapheresis, and intravenous immunoglobulin (IVIG) (reviewed in 1). Early administration of high-dose IVIGs (≤2 g/kg) is recommended for patients with TEN, even though its mechanism of action remains unclear43–45. According to a recent meta-analysis of observational studies, IVIG at dosages of ≤2 g/kg appears to significantly decrease mortality in patients with SJS or TEN43. Alternatively, cyclosporine has shown excellent efficacy for the treatment of TEN in a recent study46. However, as the supporting data for each treatment modality with regard to decreased mortality in TEN are highly controversial, further evidence based on multicenter, randomized, controlled clinical trials is still to be defined.\n\nAGEP, acute generalized exanthematous pustulosis; DRESS, drug reaction with eosinophilia and systemic reaction; MPR, maculopapular rash; SJS, Stevens-Johnson syndrome; TEN, toxic epidermal necrolysis.\n\n\nAllergologic work-up\n\nThe allergologic work-up to identify the causative agents includes skin tests (epicutaneous testing47), in vitro assays (lymphocyte transformation tests [LTTs]48–50), and drug-induced cytokine production assays (e.g. enzyme-linked immunospot [ELISpot]51). Skin tests have been shown to be safe in TEN patients, but their specificity and sensitivity are rather low51. In a recent report, Barbaud et al. performed skin patch testing to identify the causative agent in 17 patients who had suffered from SJS and/or TEN. Positive patch test reactions were observed in only 24% of those patients52. Concerning in vitro tests, it should be noted that the LTT is not a standardized procedure and merely demonstrates the proliferation of lymphocytes in the presence of various compounds. However, LTT in patients with SJS/TEN has shown low sensitivity, even if performed by highly qualified personnel53.\n\n\nConclusion\n\nSince the time TEN was first described by Dr. Lyell, it has remained a deadly disease with a mortality of around 30%. There is an unmet need to study the pathophysiology of TEN in more detail, which is complicated by the rarity of this disease and the lack of appropriate mouse models. Additionally, effective therapeutic options validated by prospective, randomized, controlled trials remain to be discovered. The most important therapeutic measure so far remains the rapid identification and withdrawal of the causative drug in addition to supportive care. However, this can be a complicated task in patients with polymedication. The allergologic work-up is further complicated by the lack of safe test methods with a high sensitivity and specificity.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nFrench LE, Prins C: Erythema multiforme, Stevens-Johnson syndrome and toxic epidermal necrolysis. In: Bolognia JL, Jorrizo JL, Schaffer JV editors. Dermatology. 3rd edition. New York: Elsevier; 2013; 319–33. Reference Source\n\nLyell A: Toxic epidermal necrolysis: an eruption resembling scalding of the skin. Br J Dermatol. 1956; 68(11): 355–61. PubMed Abstract | Publisher Full Text\n\nAchten G, Ledoux-Corbusier M: [Lyell's toxic epidermal necrolysis: histologic aspects]. Arch Belg Dermatol Syphiligr. 1970; 26(2): 97–114. PubMed Abstract\n\nRzany B, Correia O, Kelly JP, et al.: Risk of Stevens-Johnson syndrome and toxic epidermal necrolysis during first weeks of antiepileptic therapy: a case-control study. Study Group of the International Case Control Study on Severe Cutaneous Adverse Reactions. Lancet. 1999; 353(9171): 2190–4. PubMed Abstract | Publisher Full Text\n\nLa Grenade L, Lee L, Weaver J, et al.: Comparison of reporting of Stevens-Johnson syndrome and toxic epidermal necrolysis in association with selective COX-2 inhibitors. Drug Saf. 2005; 28(10): 917–24. PubMed Abstract | Publisher Full Text\n\nAguiar D, Pazo R, Durán I, et al.: Toxic epidermal necrolysis in patients receiving anticonvulsants and cranial irradiation: a risk to consider. J Neurooncol. 2004; 66(3): 345–50. PubMed Abstract | Publisher Full Text\n\nAydin F, Cokluk C, Senturk N, et al.: Stevens-Johnson syndrome in two patients treated with cranial irradiation and phenytoin. J Eur Acad Dermatol Venereol. 2006; 20(5): 588–90. PubMed Abstract | Publisher Full Text\n\nLebargy F, Wolkenstein P, Gisselbrecht M, et al.: Pulmonary complications in toxic epidermal necrolysis: a prospective clinical study. 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PubMed Abstract | Publisher Full Text\n\nBastuji-Garin S, Fouchard N, Bertocchi M, et al.: SCORTEN: a severity-of-illness score for toxic epidermal necrolysis. J Invest Dermatol. 2000; 115(2): 149–53. PubMed Abstract | Publisher Full Text\n\nYip LW, Thong BY, Lim J, et al.: Ocular manifestations and complications of Stevens-Johnson syndrome and toxic epidermal necrolysis: an Asian series. Allergy. 2007; 62(5): 527–31. PubMed Abstract | Publisher Full Text\n\nMagina S, Lisboa C, Leal V, et al.: Dermatological and ophthalmological sequels in toxic epidermal necrolysis. Dermatology. 2003; 207(1): 33–6. PubMed Abstract | Publisher Full Text\n\nBentele-Jaberg N, Guenova E, Mehra T, et al.: The Phytotherapeutic Fenugreek as Trigger of Toxic Epidermal Necrolysis. Dermatology. 2015; 231(2): 99–102. PubMed Abstract | Publisher Full Text\n\nSpielberg SP, Gordon GB, Blake DA, et al.: Predisposition to phenytoin hepatotoxicity assessed in vitro. N Engl J Med. 1981; 305(13): 722–7. PubMed Abstract | Publisher Full Text\n\nShear NH, Spielberg SP, Grant DM, et al.: Differences in metabolism of sulfonamides predisposing to idiosyncratic toxicity. Ann Intern Med. 1986; 105(2): 179–84. PubMed Abstract | Publisher Full Text\n\nWolkenstein P, Carrière V, Charue D, et al.: A slow acetylator genotype is a risk factor for sulphonamide-induced toxic epidermal necrolysis and Stevens-Johnson syndrome. Pharmacogenetics. 1995; 5(4): 255–8. PubMed Abstract\n\nDietrich A, Kawakubo Y, Rzany B, et al.: Low N-acetylating capacity in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis. Exp Dermatol. 1995; 4(5): 313–6. PubMed Abstract | Publisher Full Text\n\nYun J, Marcaida MJ, Eriksson KK, et al.: Oxypurinol directly and immediately activates the drug-specific T cells via the preferential use of HLA-B*58: 01. J Immunol. 2014; 192(7): 2984–93. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPichler WJ, Beeler A, Keller M, et al.: Pharmacological interaction of drugs with immune receptors: the p-i concept. Allergol Int. 2006; 55(1): 17–25. PubMed Abstract | Publisher Full Text\n\nAdam J, Pichler WJ, Yerly D: Delayed drug hypersensitivity: models of T-cell stimulation. Br J Clin Pharmacol. 2011; 71(5): 701–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIlling PT, Vivian JP, Dudek NL, et al.: Immune self-reactivity triggered by drug-modified HLA-peptide repertoire. Nature. 2012; 486(7404): 554–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nOstrov DA, Grant BJ, Pompeu YA, et al.: Drug hypersensitivity caused by alteration of the MHC-presented self-peptide repertoire. Proc Natl Acad Sci U S A. 2012; 109(25): 9959–64. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nYang CW, Hung SI, Juo CG, et al.: HLA-B*1502-bound peptides: implications for the pathogenesis of carbamazepine-induced Stevens-Johnson syndrome. J Allergy Clin Immunol. 2007; 120(4): 870–7. PubMed Abstract | Publisher Full Text\n\nWei CY, Chung WH, Huang HW, et al.: Direct interaction between HLA-B and carbamazepine activates T cells in patients with Stevens-Johnson syndrome. J Allergy Clin Immunol. 2012; 129(6): 1562–9.e5. PubMed Abstract | Publisher Full Text\n\nHung SI, Chung WH, Liou LB, et al.: HLA-B*5801 allele as a genetic marker for severe cutaneous adverse reactions caused by allopurinol. Proc Natl Acad Sci U S A. 2005; 102(11): 4134–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nChung WH, Hung SI, Chen YT: Human leukocyte antigens and drug hypersensitivity. Curr Opin Allergy Clin Immunol. 2007; 7(4): 317–23. PubMed Abstract | Publisher Full Text\n\nChung WH, Hung SI, Hong HS, et al.: Medical genetics: a marker for Stevens-Johnson syndrome. Nature. 2004; 428(6982): 486. PubMed Abstract | Publisher Full Text\n\nChen P, Lin JJ, Lu CS, et al.: Carbamazepine-induced toxic effects and HLA-B*1502 screening in Taiwan. N Engl J Med. 2011; 364(12): 1126–33. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nViard I, Wehrli P, Bullani R, et al.: Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin. Science. 1998; 282(5388): 490–3. PubMed Abstract | Publisher Full Text\n\nWehrli P, Viard I, Bullani R, et al.: Death receptors in cutaneous biology and disease. J Invest Dermatol. 2000; 115(2): 141–8. PubMed Abstract | Publisher Full Text\n\nIto K, Hara H, Okada T, et al.: Toxic epidermal necrolysis treated with low-dose intravenous immunoglobulin: immunohistochemical study of Fas and Fas-ligand expression. Clin Exp Dermatol. 2004; 29(6): 679–80. PubMed Abstract | Publisher Full Text\n\nChung WH, Hung SI, Yang JY, et al.: Granulysin is a key mediator for disseminated keratinocyte death in Stevens-Johnson syndrome and toxic epidermal necrolysis. Nat Med. 2008; 14(12): 1343–50. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSaito N, Qiao H, Yanagi T, et al.: An annexin A1-FPR1 interaction contributes to necroptosis of keratinocytes in severe cutaneous adverse drug reactions. Sci Transl Med. 2014; 6(245): 245ra95. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nViard-Leveugle I, Bullani RR, Meda P, et al.: Intracellular localization of keratinocyte Fas ligand explains lack of cytolytic activity under physiological conditions. J Biol Chem. 2003; 278(18): 16183–8. PubMed Abstract | Publisher Full Text\n\nTeraki Y, Kawabe M, Izaki S: Possible role of TH17 cells in the pathogenesis of Stevens-Johnson syndrome and toxic epidermal necrolysis. J Allergy Clin Immunol. 2013; 131(3): 907–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nGagliani N, Amezcua Vesely MC, Iseppon A, et al.: TH17 cells transdifferentiate into regulatory T cells during resolution of inflammation. Nature. 2015; 523(7559): 221–5. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHashizume H, Fujiyama T, Tokura Y: Reciprocal contribution of Th17 and regulatory T cells in severe drug allergy. J Dermatol Sci. 2016; 81(2): 131–4. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAng CC, Tay YK: Hematological abnormalities and the use of granulocyte-colony-stimulating factor in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis. Int J Dermatol. 2011; 50(12): 1570–8. PubMed Abstract | Publisher Full Text\n\nSchwartz RA, McDonough PH, Lee BW: Toxic epidermal necrolysis: Part II. Prognosis, sequelae, diagnosis, differential diagnosis, prevention, and treatment. J Am Acad Dermatol. 2013; 69(2): 187.e1–16; quiz 203–4. PubMed Abstract | Publisher Full Text\n\nBarron SJ, Del Vecchio MT, Aronoff SC: Intravenous immunoglobulin in the treatment of Stevens-Johnson syndrome and toxic epidermal necrolysis: a meta-analysis with meta-regression of observational studies. Int J Dermatol. 2015; 54(1): 108–15. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHuang YC, Li YC, Chen TJ: The efficacy of intravenous immunoglobulin for the treatment of toxic epidermal necrolysis: a systematic review and meta-analysis. Br J Dermatol. 2012; 167(2): 424–32. PubMed Abstract | Publisher Full Text\n\nPrins C, Kerdel FA, Padilla RS, et al.: Treatment of toxic epidermal necrolysis with high-dose intravenous immunoglobulins: multicenter retrospective analysis of 48 consecutive cases. Arch Dermatol. 2003; 139(1): 26–32. PubMed Abstract | Publisher Full Text\n\nBrockow K, Garvey LH, Aberer W, et al.: Skin test concentrations for systemically administered drugs -- an ENDA/EAACI Drug Allergy Interest Group position paper. Allergy. 2013; 68(6): 702–12. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSicherer SH, Leung DY: Advances in allergic skin disease, anaphylaxis, and hypersensitivity reactions to foods, drugs, and insects in 2014. J Allergy Clin Immunol. 2015; 135(2): 357–67. PubMed Abstract | Publisher Full Text\n\nPorebski G, Pecaric-Petkovic T, Groux-Keller M, et al.: In vitro drug causality assessment in Stevens-Johnson syndrome - alternatives for lymphocyte transformation test. Clin Exp Allergy. 2013; 43(9): 1027–37. PubMed Abstract | Publisher Full Text\n\nPolak ME, Belgi G, McGuire C, et al.: In vitro diagnostic assays are effective during the acute phase of delayed-type drug hypersensitivity reactions. Br J Dermatol. 2013; 168(3): 539–49. PubMed Abstract | Publisher Full Text\n\nRozieres A, Hennino A, Rodet K, et al.: Detection and quantification of drug-specific T cells in penicillin allergy. Allergy. 2009; 64(4): 534–42. PubMed Abstract | Publisher Full Text\n\nBarbaud A, Collet E, Milpied B, et al.: A multicentre study to determine the value and safety of drug patch tests for the three main classes of severe cutaneous adverse drug reactions. Br J Dermatol. 2013; 168(3): 555–62. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAbe R: Immunological response in Stevens-Johnson syndrome and toxic epidermal necrolysis. J Dermatol. 2015; 42(1): 42–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRive CM, Bourke J, Phillips EJ: Testing for drug hypersensitivity syndromes. Clin Biochem Rev. 2013; 34(1): 15–38. PubMed Abstract | Free Full Text"
}
|
[
{
"id": "13926",
"date": "20 May 2016",
"name": "Georg 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",
"responses": []
},
{
"id": "13927",
"date": "20 May 2016",
"name": "Areta Kowal-Vern",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13928",
"date": "20 May 2016",
"name": "Yuan-Tsong 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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-951
|
https://f1000research.com/articles/5-915/v1
|
19 May 16
|
{
"type": "Review",
"title": "Recent advances in microscopic techniques for visualizing leukocytes in vivo",
"authors": [
"Rohit Jain",
"Shweta Tikoo",
"Wolfgang Weninger",
"Shweta Tikoo"
],
"abstract": "Leukocytes are inherently motile and interactive cells. Recent advances in intravital microscopy approaches have enabled a new vista of their behavior within intact tissues in real time. This brief review summarizes the developments enabling the tracking of immune responses in vivo.",
"keywords": [
"leukocytes",
"visualizing",
"in vivo"
],
"content": "Introduction\n\nThe elicitation of an immune response against invading pathogens or tumor cells relies on a complex and highly orchestrated interplay among leukocytes, blood vessels, and stromal cell populations. During this process, immune cells need to identify and interpret structural and molecular guidance cues such as extracellular matrix fibers and chemokines to navigate through diverse tissue microenvironments towards foci of tissue injury, where they finally mediate their effector functions. Microscopic investigations into this highly orchestrated behavior of immune cells have provided insight into the workings of innate and adaptive immunity in live mice at a level of resolution previously unattained.\n\nTo visualize the behavior of immune cells in vivo, initial studies relied on bright field and epifluorescence video microscopy. Although limited in resolution and restricted by tissue clarity, these studies were instrumental for dissecting the molecular mechanisms regulating leukocyte homing during the process of extravasation1–4. However, the advent of advanced confocal5 and two-photon6,7 imaging modalities revolutionized our understanding of lymphocyte behavior within the interstitial space of intact organs, initially within re-aggregated thymic organ cultures6 and explanted intact lymph nodes5,7. These studies illuminated the complex in situ behavior of T cells within lymph nodes in the absence or presence of antigen5,7 and during their selection within the thymus6. These advancements in imaging technologies further catalyzed the emerging field of intravital imaging of the immune system. Indeed, shortly after the explant models, imaging of intact organs in situ in living mice was reported8,9. Since then, a large number of tissues/organ systems have been adapted for in vivo imaging, revealing insights into the functioning of the immune system in the steady state, during initial pathogen encounter, during the priming phase of adaptive immune responses, and within effector organs during infection, inflammation, and within tumors. The impact of intravital imaging on the field of leukocyte biology and immunology has recently been reviewed in detail elsewhere10–13.\n\nIn summary, the development of new imaging tools and molecular probes, coupled with a wealth of transgenic fluorescent reporter mice, has ushered in a new era for understanding the cellular and molecular regulators of leukocyte function in situ. In this review, we highlight some of the technical advances in this field of research, with a focus on multi-photon imaging.\n\n\nAdvances in two-photon imaging methodologies\n\nWithout doubt, the introduction of two-photon microscopy into biological imaging has enormously furthered our understanding of physiological and pathological processes at the level of intact organs. Unlike single-photon excitation used in confocal microscopy, two-photon excitation is spatially restricted to the focal plane10,12,14,15. This results in reduced levels of phototoxicity and photobleaching, enabling deep tissue imaging for prolonged periods of time10,12. Nevertheless, several technical challenges still persist. For example, physiological tissue movement due to pulsation of blood vessels, contraction of muscles, respiration, or heartbeat can be detrimental to the overall quality of the data. In addition, surgical and anesthetic requirements may alter the behavior of cells due to the induction of hypoxia, changes in tissue pH, or the generation of pro-inflammatory mediators. It is therefore imperative to develop standardized, reproducible procedures for the exposure of organs during the imaging session.\n\nFor many peripheral tissues like skin16 and draining lymph nodes8,9,17, and internal organs such as the liver18–21 and spleen22–24, tissue stabilization during imaging has been carried out using relatively simple methodologies and devices. Similarly, intravital imaging of bone marrow cavity within the skull calvaria has been adapted for two-photon microscopy25 and has played a significant role in highlighting immune cell behavior within this environment25–28. Akin to this, various methodologies have been developed for longitudinal imaging of tumors15. Cumulatively, these new imaging methodologies have provided unprecedented understanding of leukocyte behavior in vivo in a variety of tissues11–13,29–31.\n\nNonetheless, stable imaging of certain tissues, such as the brain, heart, and lung, still presents a significant challenge. In the case of the brain, the two most frequently used methodologies involve either bone thinning32,33 or removal of the cranial bone34–36 for imaging of the underlying meninges and brain parenchyma. Similarly, longitudinal imaging of the brain has been made possible by implantation of an optical window34,37,38. These methodologies allow studying the behavior of various immune cells like microglia36,39,40, monocytes41,42, and T cells13,43–46 within the intact brain and spine under homeostasis, infection, or inflammation. Newer techniques, such as implantation of glass prisms47 or micro-optical probes48, have been developed for imaging of deeper structures within the brain47,48, but they are yet to be harnessed for understanding leukocyte behavior.\n\nIn vivo imaging of lungs presents a significant technical challenge due to tissue movement and its localization within the pleural cavity. Nonetheless, several strategies have been developed that involve the closing of ventilation to the imaged lung49 or synchronizing image acquisition with respiration50. Recently, several new methodologies51–53 have described the creation of a small thoracic window, wherein the tissue is stabilized via application of a mild negative pressure51,52 or surgical glue53. These approaches maintain ventilation and perfusion within the tissue and permit imaging throughout the respiratory cycle51,52. These recent advances have highlighted the dynamic nature of T cells51,54, neutrophils28,51,53, monocytes53, macrophages55, and dendritic cells54,55 within the pulmonary tissue in the steady state and during inflammatory conditions. For instance, in vivo imaging of the lungs during influenza virus infection has identified a unique cooperative behavior between neutrophils and CD8+ effector T cells. Thus, within influenza-infected lungs, migrating neutrophils leave behind a trail of the chemokine CXCL12, which is used by effector CD8+ T cells as a guidance cue within the interstitium56. This study enforces the role of in vivo imaging in understanding the dynamic spatiotemporal cooperation between immune cells required for generating an efficient immune response.\n\nBy using endoscopic time-lapse imaging57 or optical windows58–60, researchers have recently addressed the challenges associated with in vivo imaging of leukocytes within the beating heart61. Endoscopic imaging of the heart under physiological conditions revealed the patrolling behavior of monocytes within coronary vessels57. Post-infarction, rapid recruitment of both monocytes and neutrophils was observed57,58. Surprisingly, unlike most tissues, large coronary veins but not post-capillary venules13 were the predominant sites for neutrophil extravasation post-ischemia/reperfusion injury58. Most neutrophils migrated via “hotspots” within the inflamed coronary veins58. Although this technique is still in its early days, in vivo imaging of the heart nonetheless holds immense potential in understanding leukocyte/lymphocyte behavior post-cardiac transplant and during organ rejection.\n\nIn addition to the intravital imaging of the beating heart, several methodologies have been developed to image blood vessels in vivo62–65. Studies have highlighted, for example, the dynamic migratory behavior of neutrophils63,64 and T lymphocytes63 within atherosclerotic lesions. Recently, the use of cardiac triggered acquisition coupled with image registration and post-processing image correction has provided further advancement of stable imaging of blood vessels65. This methodology enables improved temporal resolution, which has been used to define the dynamic scanning behavior of dendritic processes of leukocytes within the atherosclerotic plaques of pulsatile blood vessels65.\n\nThe use of two-photon excitation can also be exploited for inducing localized injury without physical manipulation of the surrounding tissue. This provides opportunities for studying the behavior of tissue-resident and recruited leukocytes during sterile inflammation in situ. This methodology has been used, for example, to highlight the dynamic behavior of microglia within the brain post injury36,39 and to delineate the cellular and molecular players required for interstitial migration of neutrophils66,67, monocytes67, and dendritic cells68 within the skin13,69,70. Thus, laser-induced injury has been crucial for understanding the cellular and molecular interplay required for interstitial migration of neutrophils and the damage response within the inflamed dermis66,67. Following injury, scarce initial neutrophils rapidly migrate towards the site of injury following chemokine gradients. This has been dubbed the “neutrophil scouting phase”, which is followed by an “amplification phase” wherein a large number of neutrophils display directed migration towards the injury site. This second phase is dependent on cyclic-adenosine diphosphate ribose (c-ADPR) and leukotriene B4 (LTB4) signaling66,67. The large influx of neutrophils at the injury site leads to the formation of a stable neutrophil cluster also referred to as the “stabilization phase”. The stability of the cluster is governed by signaling via G-protein-coupled receptors such as C-X-C chemokine receptor 2 (CXCR-2), N-formyl peptide receptor 2 (FPR-2), and LTB4 receptor-1 (BLT-1). Surprisingly, although integrins were dispensable for neutrophil migration within the inflamed dermis, they were required for migrating within the injury foci devoid of extracellular matrix. Together, these studies uncover the crucial role of intravital imaging in understanding the spatiotemporal dynamics of the innate immune response in vivo.\n\nThe use of in vivo imaging has also been instrumental for understanding immune responses against invading pathogens13,71,72 and has highlighted the dynamic behavior of innate and adaptive immune cells during various infections including Leishmania major73–77, Leishmania donovani78–80, Toxoplasma gondii43,81–83, Borrelia burgdorferi84,85, Staphylococcus aureus86–88, Plasmodium spp.42,46,89–93, and Mycobacterium-induced granulomas19,21. These studies have identified various strategies used by pathogens to evade immune responses; for instance, post-intradermal inoculation, Plasmodium berghei sporozoites rapidly migrate into the blood vessels and lymphatics89. In contrast, experiments performed with another vector-borne parasite, Leishmania major, revealed that these parasites do not actively migrate but rather induce neutrophil recruitment to aid parasite survival73,74,94–97. Similarly, in vivo imaging of mycobacterial granulomas within the liver have highlighted the presence of numerous antigen-presenting cells (APCs) and CD4+ T cells within the granulomatous tissue19,21. A paucity of mycobacterial antigens limited the number of stable interactions between APCs and antigen-specific CD4+ T cells and thereby suppressed the release of immunomodulatory/protective cytokines21. Cumulatively, in vivo imaging of host-pathogen interactions have identified novel cellular and molecular mechanisms involved in regulating pathogen uptake and transport, generation of adaptive immune responses, identification of new migratory strategies used for scanning infected tissues, and elicitation of effector immune responses.\n\nSimilarly, two-photon imaging of tumors has provided fascinating insights into leukocyte behavior within the tumor milieu: for example, effector CD8+ T cell-mediated scanning of tumor tissue98–100, induction of tumor cell apoptosis101, generation of T cell tolerance102, co-migration of macrophages and tumor cells103, and the role of perivascular macrophages in tumor cell dissemination104. Using experimental models that recapitulate the pathophysiology of multiple sclerosis, investigators have revealed fibrinogen-mediated perivascular clustering of microglia40, formation of immunological synapse between APCs and effector CD4+ T cells44, and neuronal cell death105 with the use of in vivo imaging.\n\nThe latest development in the use of optical phase-locked ultrasound lens provides a new methodology for high-speed volumetric imaging in vivo106. This allows for the imaging of Ca2+ signaling as well as alterations in cell morphology at high speeds106. This methodology has been used to highlight intravascular and extravascular neutrophil dynamics at a temporal resolution previously unattainable106.\n\nAltogether, the advancements in imaging instrumentation and development of new and improved physiological models have been crucial in developing a deeper and dynamic understanding of innate and adaptive immunity.\n\n\nTransgenic fluorescent reporter mouse models for in vivo imaging\n\nVisualization of cells and structures within tissues via laser excitation relies, on the one hand, on their inherent (auto)-fluorescent features; for example, second and third harmonic generation facilitate highlighting large extracellular molecules such as collagen and elastin fibers without the need for counterstaining107–110. In addition, certain auto-fluorescent metabolites such as NADPH enable the interrogation of metabolic states of cells111,112. On the other hand, most leukocyte subsets require external or genetic tagging for their detection. Initial imaging studies made use of ex vivo labeled, adoptively transferred immune cells. Although manipulation of cells may alter their behavior, these studies were crucial in understanding migratory and interactive cellular dynamics in vivo. Nevertheless, advances in genetic engineering technology coupled with the availability of a wide array of genetically encoded fluorophores have significantly expanded the scope of in vivo imaging. Introduction of transgenic fluorescent proteins at specific genetic loci or under the control of tissue- or cell-specific promoters has been crucial for following cells of interest under physiologic conditions. Numerous fluorescent reporter mouse strains are now available for in vivo imaging, and the choice of a particular reporter relies on the experimental design and the scientific question being pursued113,114. Although the detailed description of these strains is beyond the scope of this review, a few of the novel and versatile reporter mouse strains are detailed below.\n\nThe dynamic nature of immune cells presents a significant challenge while tracking the individual cell longitudinally. This challenge can be overcome by using photoactivatable fluorescent reporters, for example photoactivatable GFP115. Using such an approach, Victora and colleagues deciphered B cell116 and T follicular helper cell117 dynamics within the germinal center of murine lymph nodes, providing valuable information regarding the regulation of humoral immunity. This strategy is useful for tracking the migration and distribution of immune cells in vivo. However, the degradation of photoactivated GFP within the marked cells limits the timespan available for tracking, usually in the order of days (t½ - 30 hours for naive B cells116). More recently, photo-switchable transgenic reporters like KikGR118,119 and Kaede120 mice have become available. Briefly, in Kaede mice, ubiquitously expressed photoconvertible GFP121 undergoes peptide cleavage and alteration within the chromophore upon exposure to ultraviolet light, resulting in a shift from green to red fluorescence120. This conversion is irreversible and thereby marks the treated cell until the photoconverted Kaede is degraded and replaced by new Kaede protein120. Kaede mice have been used to decipher the migratory kinetics and behavior of T follicular helper cells under homeostasis or during memory responses122. In brief, the above-mentioned strategies coupled with in vivo imaging have revealed the dynamic behavior of T follicular helper cells during primary and secondary immune responses. During the primary response, T follicular helper cells are restricted within the germinal center, where they provide cognate help to developing B cells. However, during secondary immune responses (akin to a recurrent infection), the memory T follicular helper cells show unrestricted migration in and out of the germinal centers, a strategy that might be useful in eliciting rapid humoral responses122.\n\nAlthough both KikGR and Kaede mice have been used for tracking the dynamics of various immune cell subsets like neutrophils123, T cells124,125, innate lymphoid cells126, and dendritic cells127 at a population level, great opportunities still persist in understanding the behavior of these immune cells at single-cell resolution.\n\nAnother strategy to irreversibly mark individual cells in vivo relies on the Cre/lox recombination system. Using this strategy, Livet and colleagues generated the Brainbow mouse strain where Cre-mediated recombination stochastically permutates multiple copies of the construct containing several different fluorescent proteins128,129. This recombination strategy results in a mosaic of fluorescent colors leading to the detection of nearly 90 separate colors that can be used to mark individual cells. This strategy was initially used to identify individual neurons within the CNS and for tracing neuronal circuitry128. The generation of the Brainbow mouse paved the way for the development of several new approaches like LeGO130, Confetti131, and Ubow132 to mark cells for tracing the fate of individual cells. Ubow mice have been used for fate mapping of Langerhans cells132 and follicular dendritic cells133 within the skin and lymph node, respectively. Recently, a combination of fluorescent reporters and the Cre/lox system has been used exquisitely to highlight in vivo transfer of metastatic information between tumor cells134. Use of the Cre/lox system provides a significant advantage over other strategies such as photoactivation or photoconversion by genetically/permanently labeling the cells, thereby enabling tracking throughout the lifetime of the cell. However, in most scenarios, the genetic labeling is targeted to a subset of cells and not to spatiotemporally selected individual cells.\n\nTaken together, the recent advances in recombinant DNA technology like TALEN and the CRISPR/Cas9 genome editing system along with the availability of a multitude of reporter and knockout mouse strains promise a more colorful and dynamic future for the in vivo imaging of leukocytes.\n\n\nOther methodologies\n\nAlthough the use of transgenic fluorescent reporter strains for in vivo imaging has been gaining momentum, the transduction of immune cells using various fluorescent reporters still provides a valuable tool for understanding leukocyte function. These strategies have been used to delineate activation45,102,135, calcium signaling136, and apoptosis137 within T cells in vivo. Similarly, several fluorescent probes are available to highlight cellular and molecular events crucial for leukocyte function in vivo. These fluorescent probes have been utilized to decipher various cellular processes: for example, NETosis88,138, the generation of reactive oxygen species139, and cell death67,140. The use of fluorescent probes to mark DNA coupled with in vivo imaging has been crucial in deciphering the physiological role of DNA-NETs (DNA-neutrophil extracellular traps). Intravital imaging in various infectious model systems has uncovered the important role of DNA-NETs in ensnaring and killing pathogens88,141–143. Although DNA-NETs are an essential arsenal of the innate immune response, release of DNA-NETs in vivo can trap circulating tumor cells and enhance metastasis144.\n\nLikewise, novel approaches have been developed to couple immune cell behavior with receptor-based signaling in vivo via administration of labeled antibodies. In this methodology, labeled Fab fragments are administered in vivo and the distribution of cell surface receptors and the behavior of labeled cells are recorded, segmented, and analyzed as flow cytometry plots145. This methodology is of immense importance where the migratory dynamics of immune cells need to be analyzed in context with the receptor signals delivered to individual immune cells in situ.\n\n\nConcluding remarks\n\nIn the past decade, in vivo two-photon imaging has provided researchers with unparalleled views of immune cell behavior under homeostasis or pathology. This has resulted in the identification of new molecular and cellular regulators of the immune system.\n\nIn our laboratory, in vivo imaging has been instrumental in highlighting the dynamic behavior of neutrophils66, monocytes42, T cells146,147, dendritic cells73, type-2 innate lymphoid cells148, and perivascular macrophages86 under homeostasis or during various infections or inflammatory conditions.\n\nNonetheless, the use of in vivo imaging to understand cell-to-cell communication and the generation of complex interaction networks still requires extensive investigation. Intravital multiphoton imaging in most tissues is restricted to approximately 500 μm from the surface, which limits the visualization of processes within deeper tissues in vivo, therefore leaving certain aspects of the immune response in the dark. Various other strategies such as optical frequency domain imaging (OFDI)149 and speckle-variance optical coherence tomography (svOCT)150 have been developed for overcoming this limitation; however, these methodologies currently lack the necessary resolution for imaging immune cell behavior and are mostly useful for imaging tissue architecture and the vasculature151,152.\n\nWe believe that the increased availability of fluorescent reporters for in vivo marking of cellular and molecular signaling, technical improvements in instrumentation (for example, adaptive optics), and better analytical capability will be crucial for understanding the complex behavior of leukocytes in vivo.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nWolfgang Weninger was supported by project grants 1066477, 1102398, 1104876 and 1106439, from the National Health and Medical Research Council Australia. Rohit Jain was supported by an Early Career Researcher grant from the Sydney Medical School, Sydney, Australia. Shweta Tikoo was supported by an Early Career Researcher grant from the Sydney Medical School and PdCCRS grant from Cure Cancer Australia.\n\n\nReferences\n\nButcher EC, Picker LJ: Lymphocyte homing and homeostasis. Science. 1996; 272(5258): 60–6. PubMed Abstract | Publisher Full Text\n\nSchmidt EE, MacDonald IC, Groom AC: Interactions of leukocytes with vessel walls and with other blood cells, studied by high-resolution intravital videomicroscopy of spleen. Microvasc Res. 1990; 40(1): 99–117. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndo R, Hama H, Yamamoto-Hino M, et al.: An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci U S A. 2002; 99(20): 12651–6. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSuan D, Nguyen A, Moran I, et al.: T follicular helper cells have distinct modes of migration and molecular signatures in naive and memory immune responses. Immunity. 2015; 42(4): 704–18. PubMed Abstract | Publisher Full Text\n\nHampton HR, Bailey J, Tomura M, et al.: Microbe-dependent lymphatic migration of neutrophils modulates lymphocyte proliferation in lymph nodes. Nat Commun. 2015; 6: 7139. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nTomura M, Honda T, Tanizaki H, et al.: Activated regulatory T cells are the major T cell type emigrating from the skin during a cutaneous immune response in mice. 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}
|
[
{
"id": "13908",
"date": "19 May 2016",
"name": "Tim Lammermann",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13909",
"date": "19 May 2016",
"name": "Peter Friedl",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13910",
"date": "19 May 2016",
"name": "Tri Phan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-915
|
https://f1000research.com/articles/5-914/v1
|
19 May 16
|
{
"type": "Research Article",
"title": "The GenABEL Project for statistical genomics",
"authors": [
"Lennart C. Karssen",
"Cornelia M. van Duijn",
"Yurii S. Aulchenko",
"Cornelia M. van Duijn"
],
"abstract": "Development of free/libre open source software is usually done by a community of people with an interest in the tool. For scientific software, however, this is less often the case. Most scientific software is written by only a few authors, often a student working on a thesis. Once the paper describing the tool has been published, the tool is no longer developed further and is left to its own device. Here we describe the broad, multidisciplinary community we formed around a set of tools for statistical genomics. The GenABEL project for statistical omics actively promotes open interdisciplinary development of statistical methodology and its implementation in efficient and user-friendly software under an open source licence. The software tools developed withing the project collectively make up the GenABEL suite, which currently consists of eleven tools. The open framework of the project actively encourages involvement of the community in all stages, from formulation of methodological ideas to application of software to specific data sets. A web forum is used to channel user questions and discussions, further promoting the use of the GenABEL suite. Developer discussions take place on a dedicated mailing list, and development is further supported by robust development practices including use of public version control, code review and continuous integration. Use of this open science model attracts contributions from users and developers outside the “core team”, facilitating agile statistical omics methodology development and fast dissemination.",
"keywords": [
"Open source",
"Scientific software",
"Software development",
"Community building",
"Statistical genetics",
"Genomics",
"Statistical methodology"
],
"content": "Introduction\n\nThe field of statistical (gen-)omics lies at the heart of current research into the genetic aetiology of (human) disease and personalized or precision medicine1. Genome-wide association studies (GWAS), genotype imputation and next-generation sequencing (NGS) are just a few of the techniques used in this field that is driven by increasingly larger data sets2,3. With the advent of polyphenotype analysis as is now customary in e.g. lipidomics and metabolomics, the issues of dealing with big data have become imminent4,5. In recent years, scientists and funding organizations alike have come to realize that in order to successfully tackle the challenges of the field, close collaboration between various disciplines, e.g. statistics, molecular biology, genetics, and computer science, is of paramount importance2,6,7.\n\nMany software tools developed by scientists are distributed as free/libre open source software (FLOSS). FLOSS tools are often developed by groups of people with different backgrounds, working from different geographical locations, either under central guidance or in a loose cooperation, sometimes as part of their employment, sometimes “just for fun”8. The key to successful, sustainable open source software is an active community of both developers/contributors and end users9. Unfortunately, creators of scientific software are usually not funded to actively build such a community. Moreover, our experience shows that once the peer-reviewed article describing a tool has been published, funding and time to continue development and support of that tool are usually limited or non-existent, and consequently, the tool often slowly fades into oblivion. It needs no explanation that this amounts to a waste of effort and money.\n\nIt was with these premises in mind that the GenABEL Project for Statistical Genomics was started as an extension of the original community of users and developers around the GenABEL package10.\n\n\nThe GenABEL project\n\nThe GenABEL project aims to provide a framework for collaborative, sustainable, robust, transparent, opensource based development of statistical genomics methodology. Within the project, statisticians devoted to method development work together with statistical geneticists and biologists to refine existing statistical methods as well as develop new ones and make them applicable to genomic analysis. With the help of computer scientists and scientific software developers these mathematical models are then implemented into efficient and user-friendly software. This flow of work and information is not linear, but rather more circular in nature, with information and feedback being continuously transferred between the various layers as depicted in Figure 1. In short, it is a form of agile community-driven development11,12.\n\nOpenness is an important aspect of the GenABEL project13. It enables a free flow of information between the layers in the project resulting in rapid feedback between the various levels. Not only do we require that all tools are released under an open source or free software licence like the GNU Public Licence (GPL), we also try to create an atmosphere of open communication using public mailing lists and web forums (see the sections Interaction with the user community and Development infrastructure below). Moreover, because of this openness results of the project (i.e. statistical methods as well as software packages) are easily disseminated among the end users, be they epidemiologists, bioinformaticians or others.\n\n\nThe GenABEL suite\n\nThe software tools developed within the GenABEL project collectively make up the GenABEL suite. Many tools are R packages, however, this is not a requirement for inclusion in the suite. Any software that is related to the field of statistical (gen-)omics is welcome (technical requirements are discussed in section Development infrastructure). Currently, the suite consists of 11 officially released tools (cf. Table 1) and two that are in beta stage.\n\nCurrently, all tools are licensed under the GNU Public Licence (GPL).\n\nThe GenABEL R package (not to be confused with the GenABEL project or the GenABEL suite), provides an efficient file format for storing genotype data and facilitates pre-GWAS quality control as well as running GWAS of continuous and binary phenotypes, and time-to-event data. The collaborative nature of the project is demonstrated in the GenABEL package as it implements several statistical methods developed within the framework, including approximate mixed models21–23 and various methods for genomic control24,25. This shows that the project is really a platform for implementation of (statistical) methods which removes the burden of thinking about data formats etc. allowing method developers to focus on what they do best. The GenABEL package is the most popular package in the GenABEL suite with more than 809 citations of its paper (according to Google Scholar)10.\n\nProbABEL is a tool for running GWAS on imputed genotype data. Like the GenABEL package it allows running linear or logistic regression, as well as Cox proportional hazards model, however, ProbABEL is tailored to the large file sizes that are inherent to current data sets with approximately 30 million imputed genotypes per individual. It is the second most-used tool from the suite with more than 267 citations (according to Google Scholar)14.\n\nAs indicated by its name, MixABEL is an R package for running genome-wide association analyses using mixed models in quantitative traits.\n\nGWAS usually involves meta-analysis of the regression results of various cohorts. The R package MetABEL provides simple meta-analysis functions including generation of forest plots.\n\nThe R package VariABEL can be used to look for variance heterogeneity in genetic studies. Such heterogeneity is an indication of interaction between a genetic marker and either another marker or an unknown factor16,26.\n\nIn 2013 OmicABEL was added to the suite. It contains a high-performance computing based approach facilitating extremely fast mixed-model based regression of multiple omics traits like metabolomics or lipidomics on imputed genotype data18. OmicABEL aims to increase computational throughput while reducing memory usage and energy consumption. This was achieved by using optimal (hardware-tailored) algorithms using state-of-the-art linear algebra kernels, incorporating optimizations and avoiding redundant computations.\n\nPredictABEL is an R package for the assessment of genetic risk prediction models. It includes functions to compute univariate and multivariate odds ratios of the predictors, the area under the receiver operating characteristic (ROC) curve (AUC), Hosmer-Lemeshow goodness of fit test, reclassification table, net reclassification improvement and integrated discrimination improvement17.\n\nRepeatABEL allows one to run a GWAS for multiple observations on related individuals19. Like ParallABEL, this package is a great example of contributions by the community since its development was not initiated by the core GenABEL developers.\n\nCollapsABEL is the most recent addition to the GenABEL suite. It is an R library for detecting compound heterozygote (CH) alleles in GWAS. It is a computationally efficient solution for screening general forms of CH alleles in densely imputed microarray or whole genome sequencing datasets20.\n\nApart from the aforementioned packages which directly address certain types of analysis and/or data management, several packages in the suite have a supportive role. DatABEL is an R interface to our filevector library which provides a file format that is optimised for fast access to data in matrix form, e.g. imputed genotype data. ParallABEL is an R library for parallel execution of GWAS in R.\n\nThe latest stable version of the R packages are available on CRAN (http://cran.r-project.org), the Comprehensive R Archive Network. The source code for the other packages can be downloaded from our website at http://www.genabel.org, from the project’s version control server or on GitHub (see section Development infrastructure).\n\n\nInteraction with the user community\n\nThe GenABEL project website is the central hub that points to package descriptions, tutorials, the development website, and other information for potential and existing users and developers. Usage statistics such as number of visits and country of origin of visitors are monitored using Google Analytics (http://www.google.com/analytics/) in order to get an estimate of the number of users of the tools and their origins. As an example of the information that can be obtained from this data, Figure 2 shows the top 20 cities of origin of the visitors of the GenABEL website in the period of 28 April 2015 till 28 April 2016. Only visits lasting more than 60 seconds and cities with more than 15 visits were taken into account in an attempt to filter out “accidental” visits. The website was visited 16319 times in that period, of which 696 visits were from an unknown city.\n\nOnly visits lasting more than 60 seconds and from cities from which more than 15 visits originated were taken into account. The total number of visits in that period was 16319, of which 696 came from unknown cities. Each city name is followed by the two-letter ISO code of the country in which it is located.\n\nCollecting visitor data like this helps getting an insight in the institutes that use software from the GenABEL suite, which can then be used to show the impact the tools have, e.g. when applying for funding.\n\nInteraction with the user community is done via social media like Twitter (https://twitter.com/GenAproj) and Facebook (https://www.facebook.com/pages/GenABEL-project/329281857167394), as well as a dedicated mailing list for announcements of new package releases, making it easy for both users and system administrators to keep up to date with new releases and developments in the GenABEL project and the GenABEL suite.\n\nEach tool in the GenABEL suite has its own documentation and the GenABEL Tutorial27 with more than 260 pages takes the user from learning basic R to performing more complicated analyses, showing how the various packages interconnect. Moreover, several video tutorials are available online.\n\nInteractive user support is mostly done through our forum (http://forum.genabel.org). Having an open forum serves various purposes. First of all it is a central, easy to point to reference. Moreover, compared to having individual users e-mailing a package author, who may be on holiday or otherwise unavailable, an open forum where users and developers collaborate helps in shortening the time-to-answer. Furthermore, having an active forum where users can help each other allows the developers to focus on fixing bugs and implementing new features. As of March 1st, 2015, the GenABEL forum has 538 activated user accounts, with an average 2.92 new registrations per week since the start of the forum in January 2011. These users have contributed 1422 posts in 427 topics, with an average 7.15 posts per week.\n\nThe first hurdle many users of (scientific) software encounter is the installation process. Within the GenABEL project we aim to make installation as simple as possible. Using CRAN for the R packages makes installation and upgrading as simple as typing a single command. For the tools that don’t use R, we aim to provide up-to-date packages for various Linux distributions. Currently, ProbABEL is packaged in the Stable, Testing and Unstable repositories of Debian with the help of the Debian Med team28. Other packages are planned to be added before the end of 2016. For Ubuntu Linux a Personal Package Archive is available (https://launchpad.net/~l.c.karssen/+archive/genabel-ppa). Packages for Red Hat Enterprise Linux and CentOS are on the road map, but haven’t been released yet.\n\n\nDevelopment infrastructure\n\nThe GenABEL project welcomes contributions of all sorts, from new tools to fixing spelling errors in the documentation, to bug reports and feature requests. To this end all program code and documentation are either stored in a publicly readable instance of the Subversion version control system, with write access limited to a group of core contributors, or on GitHub (https://github.com/GenABEL-Project), which is one of the leading platforms for what is termed “social coding”, which perfectly fits the project’s goals. These version control systems record any change to the files so they can easily be reviewed and reverted if necessary7,29,30.\n\nIn November 2010 a mailing list was created as a central place for development discussions. As of April 2016 this list has 34 subscribers. The GenABEL development website (https://r-forge.r-project.org/projects/genabel/) including the Subversion server, the mailing lists, and the trackers for bugs and feature requests are kindly provided by the R-Forge project31. Currently, a total of 94 bugs have been submitted to the bug trackers on R-forge and GitHub since their opening in 2010 and 2015, respectively. Of these 94, 12 were directly contributed by people outside of the core team of developers. Another 42 bug reports were filed by regular contributors based on user reports on the forum, for example, which means that 56% of the bugs have been reported by people in our community that are not core contributors.\n\nIn order to be able to maintain the quality of both old and new software in the GenABEL suite prospective tools go through a review process in which both the functional quality of the code is evaluated (does the tool do what it intends to do?), as well as the actual quality of the code (is the code clearly written, including developer documentation in the form of e.g. comments; does the code conform to the GenABEL coding style guidelines; etc.). Moreover, as set out in the GenABEL developer guidelines, we expect commitment of the person or team submitting a tool to the suite to maintain and support it, otherwise the maintenance burden would end up with the core team and it would be too easy to create a tool, write a paper and then ‘dump’ it in the GenABEL project hoping “the community” will take care of it. Therefore, the community has the option to mark a tool as obsolete, warning the user that bugs will no longer be fixed and support is limited or non-existent.\n\nIn 2013 we have started to use a Jenkins Continuous Integration server. Using Jenkins various tests (e.g. regression tests, build tests and tests for memory leaks) are automatically run on each commit to the version control systems. Consequently, changes that break existing functionality are detected at an early stage, thus leading to more stable software releases.\n\n\nConclusion\n\nThe original publication of the GenABEL package for statistical analysis of genotype data10 has led to the evolution of a community which we now call the GenABEL project, which brings together scientists, software developers and end users with the central goal of making statistical genomics work by openly developing and subsequently implementing statistical models into user-friendly software.\n\nThe project has benefited from an open development model, facilitating communication and code sharing between the parties involved. The use of a free software licence for the tools in the GenABEL suite promotes quick uptake and widespread dissemination of new methodologies and tools. Moreover, public access to the source code is an important ingredient for active participation by people from outside the core development team and is paramount for reproducible research. Feedback from end users is actively encouraged through a web forum, which steadily grows into a knowledge base with a multitude of answered questions. Furthermore, our open development process has resulted in transparent development of methods and software, including public code review, a large fraction of bugs being submitted by members of the community, and quick incorporation of bug fixes.\n\n\nData and software availability\n\nThe file tracker_report-2016-04-16.csv contains the data exported from the GenABEL R-forge bug tracker as it was on the date listed in the file name. Because of the recent move of some of the tools from R-forge to Github, the number of issues on the Github pages of the GenABEL project was still low. Therefore, these were counted manually.\n\nThe file Analytics www.genabel.org Locatie Lennart 20150428-20160428.csv contains the data extracted from the Google Analytics page for the GenABEL website for the period listed in the file name. The columns contain the ISO code of the country, city, number of sessions, number of new viewers, bounce percentage, pages per session and average session duration, respectively.\n\nThe file analysis_GenABELpaper.org contains the source code used for the automated data extraction for this paper in Emacs Org mode literate programming format (http://orgmode.org)32.\n\nThe code contained in the Org mode file and the data in the csv files listed above are in the public domain (Creative Commons CC0 license) and can be used without restriction.\n\nThe data related to the GenABEL forum were extracted manually from the forum control panel.\n\nThe tools currently in the GenABEL suite are all Free Software, licensed under the GNU Public License. An up-to-date list of the packages in the suite can be found on http://www.genabel.org/packages, which also contains pointers to the source code of the latest stable versions and the version control repositories on R-forge and GitHub (see the section Development infrastructure above for the URLs).\n\nArchived source code at the time of publication https://zenodo.org/record/5100833",
"appendix": "Author contributions\n\n\n\nCMvD and YSA jointly conceived the GenABEL suite. YSA conceived the idea the GenABEL project and formulated its initial guidelines. LCK and YSA are co-authors and maintainers of various packages in the GenABEL suite and act as maintainers of parts of the project’s infrastructure (e.g. forum and mailing lists). LCK drafted the initial version of the manuscript and analyzed the data. All authors contributed to the review of the manuscript and agreed to the final content.\n\n\nCompeting interests\n\n\n\nThe authors declare no conflicts of interest in the authorship or publication of this contribution.\n\n\nGrant information\n\nFunding from the following sources enabled work on specific packages in the GenABEL suite: PolyOmica, Groningen, The Netherlands; Centre for Medical Systems Biology (CMSB), The Netherlands; the Netherlands Genomics Initiative (NGI); the Netherlands Organisation for Scientific Research (NWO); the Department for Health Evidence, Radboud University Medical Centre, Nijmegen, The Netherlands; Deutsche Forschungsgemeinschaft (German Research Association, grant GSC 111); Russian Foundation for Basic Research (RFBR, 12-04-33182, 15-34-20763, 15-04-07874); the RFBR-Helmholtz society Joint Research Groups programme (12-04-91322); the European Union FP7 framework projects MIMOmics (grant agreement nr. 305280) and Pain-Omics (grant agreement nr. 602736). The work of YSA was supported by a grant from the Russian Science Foundation (RSCF 14-14-00313).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank the GenABEL community, i.e. the people who have used the software, filed bug reports, posted on the forum, or contributed tools, patches or ideas, for their involvement.\n\n\nReferences\n\nCollins FS, Varmus H: A new initiative on precision medicine. N Engl J Med. 2015; 372(9): 793–795. PubMed Abstract | Publisher Full Text\n\nMargolis R, Derr L, Dunn M, et al.: The National Institutes of Health’s Big Data to Knowledge (BD2K) initiative: capitalizing on biomedical big data. J Am Med Inform Assoc. 2014; 21(6): 957–958. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarx V: Biology: The big challenges of big data. Nature. 2013; 498(7453): 255–260. PubMed Abstract | Publisher Full Text\n\nDemirkan A, Henneman P, Verhoeven A, et al.: Insight in genome-wide association of metabolite quantitative traits by exome sequence analyses. PLoS Genet. 2015; 11(1): e1004835. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDemirkan A, van Duijn CM, Ugocsai P, et al.: Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations. PLoS Genet. 2012; 8(2): e1002490. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnapp B, Bardenet R, Bernabeu MO, et al.: Ten simple rules for a successful cross-disciplinary collaboration. PLoS Comput Biol. 2015; 11(4): e1004214. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMerali Z: Computational science: ...Error. Nature. 2010; 467(7317): 775–777. PubMed Abstract | Publisher Full Text\n\nTorvalds L, Diamond D: Just for Fun: The Story of an Accidental Revolutionary. HarperBusiness, 2002. Reference Source\n\nFogel K: Producing Open Source Software: How to Run a Successful Free Software Project. O’Reilly Media, first edition, 2005; ISBN: 978–0–596–00759–1. Reference Source\n\nAulchenko YS, Ripke S, Isaacs A, et al.: GenABEL: an R library for genome-wide association analysis. Bioinformatics. 2007; 23(10): 1294–1296. PubMed Abstract | Publisher Full Text\n\nKane DW, Hohman MM, Cerami EG, et al.: Agile methods in biomedical software development: a multi-site experience report. BMC Bioinformatics. 2006; 7: 273. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBudd A, Corpas M, Brazas MD, et al.: A quick guide for building a successful bioinformatics community. PLoS Comput Biol. 2015; 11(2): e1003972. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrli´c A, Procter JB: Ten simple rules for the open development of scientific software. PLoS Comput Biol. 2012; 8(12): e1002802. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAulchenko YS, Struchalin MV, van Duijn CM: ProbABEL package for genome-wide association analysis of imputed data. BMC Bioinformatics. 2010; 11: 134. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSangket U, Mahasirimongkol S, Chantratita W, et al.: ParallABEL: an R library for generalized parallelization of genome-wide association studies. BMC Bioinformatics. 2010; 11: 217. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStruchalin MV, Amin N, Eilers PH, et al.: An R package \"VariABEL\" for genome-wide searching of potentially interacting loci by testing genotypic variance heterogeneity. BMC Genet. 2012; 13: 4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKundu S, Aulchenko YS, van Duijn CM, et al.: PredictABEL: an R package for the assessment of risk prediction models. Eur J Epidemiol. 2011; 26(4): 261–264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFabregat-Traver D, Sharapov SZh, Hayward C, et al.: High-performance mixed models based genome-wide association analysis with omicABEL software [version 1; referees: 2 approved, 1 approved with reservations]. F1000Res. 2014; 3: 200. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRönnegård L, McFarlane ES, Husby A, et al.: Increasing the power of genome wide association studies in natural populations using repeated measures-evaluation and implementation. Methods Ecol Evol. 2016. Publisher Full Text\n\nZhong K, Karssen LC, Kayser M, et al.: CollapsABEL: an R library for detecting compound heterozygote alleles in genome-wide association studies. BMC Bioinformatics. 2016; 17(1): 156. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAulchenko YS, de Koning D, Haley C: Genomewide rapid association using mixed model and regression: a fast and simple method for genomewide pedigree-based quantitative trait loci association analysis. Genetics. 2007; 177(1): 577–585. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSvishcheva GR, Axenovich TI, Belonogova NM, et al.: Rapid variance components-based method for whole-genome association analysis. Nat Genet. 2012; 44(10): 1166–1170. PubMed Abstract | Publisher Full Text\n\nBelonogova NM, Svishcheva GR, van Duijn CM, et al.: Region-based association analysis of human quantitative traits in related individuals. PLoS One. 2013; 8(6): e65395. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmin N, van Duijn CM, Aulchenko YS: A genomic background based method for association analysis in related individuals. PLoS One. 2007; 2(12): e1274. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTsepilov YA, Ried JS, Strauch K, et al.: Development and application of genomic control methods for genome-wide association studies using non-additive models. PLoS One. 2013; 8(12): e81431. 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\nAulchenko YS, Karssen LC, The GenABEL project developers: The GenABEL Tutorial. Zenodo. 2015. Publisher Full Text\n\nMöller S, Krabbenhöft HN, Tille A, et al.: Community-driven computational biology with Debian Linux. BMC Bioinformatics. 2010; 11(Suppl 12): S5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilson G, Aruliah DA, Brown CT, et al.: Best practices for scientific computing. PLoS Biol. 2014; 12(1): e1001745. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlischak JD, Davenport ER, Wilson G: A Quick Introduction to Version Control with Git and GitHub. PLoS Comput Biol. 2016; 12(1): e1004668. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTheußl S, Zeileis A: Collaborative software development using R-forge. R J. 2009; 1(1): 9–14. Reference Source\n\nSchulte E, Davison D, Dye T, et al.: A multi-language computing environment for literate programming and reproducible research. J Stat Softw. 2012; 46(3): 1–24. Reference Source\n\nKarssen LC, van Duijn CM, Aulchenko YS: Data of GenABEL Project for Statistical Genomics. 2016. Data Source"
}
|
[
{
"id": "14057",
"date": "27 May 2016",
"name": "Giulietta Minozzi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis very well written article describes the GenABEL project for statistical genomics and high lightens the great success of the project, that in the years has lead to the creation of an actual scientific community that is spread in several countries worldwide.\n\nIt is my understanding that the manuscript is aimed at reaching potential new users, but even to old users, unaware of the possible new tools included in the GenABEL project.\n\nThe methodologies underlining the different tools and their potential applications are well described. Figure 2 describes the extent of the use of the GenABEL website/suite and Table 1 gives a glance of the tools available and on the new versions implemented.\n\nIt is clear, from the texts, the effort that has taken place and is taking place aiming at bringing together top scientists, software developers and end users with the central goal of making statistical genomics work by openly developing and subsequently implementing statistical models into a user-friendly software.\n\nI retain this article, the tools and the information provided of extreme importance to the scientific community.",
"responses": []
},
{
"id": "14125",
"date": "17 Jun 2016",
"name": "Bjarni J. Vilhjálmsson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nKarsen et al. presents a paper on the GenABEL project, which is really a software suite that contains 11 different packages. This is a large and impressive project that contains packages that are routinely used by researchers studying genetics. Indeed, this is reflected by more than 800 citations (according to google scholar) for the original GenABEL paper published in 2006. However, the 11 packages presented in this paper have been published previously in some shape or form, albeit (presumably) not in their most recent version. It is therefore tempting to question the novelty of the current paper that summarizes the GenABEL project and describes its user and developer community. Nevertheless, despite limited amount of novel scientific ideas or scientific results in the current manuscript, the authors have clearly put a lot of work into creating a very impressive interactive user and developer community. Furthermore, the paper is well written and it will undoubtedly be highly cited by future researchers. Lastly, to reiterate, the GenABEL is a very impressive large scale project that is heavily used by the community! I therefore think this is overall a nice publication that is certainly suitable for indexation.\n\nMinor comments:\nAbstract: “…developed withing..” -> “…developed within..”\n\nI think it would be magnanimous if you acknowledged contributors as authors, e.g. under an umbrella term “The GenABEL community”.\n\nLeave citations counts out of manuscript, it looks awkward. They will also change.\n\nOn p. 3 “polyphenotype”, I think multivariate would be a better word.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-914
|
https://f1000research.com/articles/5-912/v1
|
19 May 16
|
{
"type": "Review",
"title": "The potential of plants as a system for the development and production of human biologics",
"authors": [
"Qiang Chen",
"Keith R. Davis",
"Qiang Chen"
],
"abstract": "The growing promise of plant-made biologics is highlighted by the success story of ZMapp™ as a potentially life-saving drug during the Ebola outbreak of 2014-2016. Current plant expression platforms offer features beyond the traditional advantages of low cost, high scalability, increased safety, and eukaryotic protein modification. Novel transient expression vectors have been developed that allow the production of vaccines and therapeutics at unprecedented speed to control potential pandemics or bioterrorism attacks. Plant-host engineering provides a method for producing proteins with unique and uniform mammalian post-translational modifications, providing opportunities to develop biologics with increased efficacy relative to their mammalian cell-produced counterparts. Recent demonstrations that plant-made proteins can function as biocontrol agents of foodborne pathogens further exemplify the potential utility of plant-based protein production. However, resolving the technical and regulatory challenges of commercial-scale production, garnering acceptance from large pharmaceutical companies, and obtaining U.S. Food and Drug Administration approval for several major classes of biologics are essential steps to fulfilling the untapped potential of this technology.",
"keywords": [
"plant-made biologics",
"plant-host engineering",
"plant expression platforms"
],
"content": "Introduction\n\nA significant chapter in the 2014–2016 Ebola outbreak came from the survival of two infected American health aid workers. Dr Kent Brantly and Nancy Writebol’s Ebola infection quickly escalated to the point where they thought they were dying. Remarkably, their condition dramatically improved soon after receiving an experimental drug called ZMapp™1. ZMapp™ is a cocktail of three monoclonal antibodies (mAbs) produced in plants (Figure 1). The fact that plant-produced proteins can be life-saving drugs has brought renewed attention to the field of plant-made biologics (PMBs). Why are plants used to produce ZMapp™ and other biologics? Will plant-based expression systems outcompete mammalian cell culture systems as a general platform for biologics production in the future? What are the remaining challenges that have to be overcome for this technology to fulfill its potential? These are some of the questions that have been repeatedly asked by the general public and drug development strategists. In this review, we aim to address these questions.\n\n\nWhy use plants?\n\nProtein-based biologics comprise the largest and fastest growing class of pharmaceutical products. Currently, the majority of human biologics are produced in mammalian and microbial cell cultures. Biologics produced in cell cultures require capital-intensive facilities, fermenters, expensive downstream processing, cold storage and transportation, and sterile delivery methods. These limitations encourage the development of alternative production systems.\n\nIn contrast to animal- and microbial-based cell culture platforms, early studies of PMBs emphasized the advantages of plants with respect to their low production costs, high scalability in upstream protein expression, and increased safety2. Given that plants rarely carry human or animal pathogens, the risk of introducing pathogens is far lower compared to mammalian cell production. Another advantage of biologic production in plants is that it does not require capital-prohibitive facilities, bioreactors, and expensive culture media but can be easily scaled in relatively inexpensive greenhouses with simple mineral solutions. Thus, lower manufacturing costs have been widely assumed as an innate advantage of plant-based production platforms.\n\nIt is crucial to understand the true cost of PMBs because manufacturing cost does have an impact on the market acceptability and profitability of a product. This has been a controversial topic, as information on the actual costs of producing PMBs at industrial scale has not been readily available. Recent case studies by Tusé et al. provide urgently needed economic evaluations of the current PMB platforms. Their studies revealed that plant-based platforms can substantially reduce the production cost of biologics compared with traditional platforms, with upstream costs of goods as low as $1.00–2.00 per kilogram of protein3. However, it is important to note that the cost of downstream processing for PMBs, especially for parenteral applications, is estimated to be similar to that of other production systems. These studies provide the first direct evidence to support the long-held belief that plants can lower the cost of biologic production.\n\nNew plant expression systems also offer the flexibility and speed that cannot be matched by production technologies based on mammalian cell culture. This is due to innovations in expression-vector development, particularly vectors for transient expression. The development of ‘deconstructed’ viral vector systems (e.g. magnICON, geminiviral, and pEAQ) has successfully addressed the challenges of insufficient protein expression levels, consistency, and speed of biologic production in plants4–6. For example, transient expression with deconstructed viral vectors allows the production of up to 5 milligrams of mAb per gram of fresh leaf weight within 2 weeks, in contrast to using a process that requires generating and selecting transgenic plants; this can take from months to years. Moreover, transgenic plant production often results in low and inconsistent protein yield7. The rapid and high-level protein production capability of transient expression technology makes it the optimal system to produce milligram and gram levels of biologics for pre-clinical studies. ‘Bridge’ versions of these vectors have also been developed for scale-up manufacturing of biologics in stable transgenic plants8,9. Thus, ‘deconstructed’ viral vectors offer a set of versatile tools that can rapidly evaluate biologic candidates and then transition them to large-scale commercial manufacturing.\n\nHost engineering is another source of innovation that equips plant-expression systems with additional advantages. For example, the minor differences in N-glycosylation between plant and human cells have been a major concern, as they may trigger the production of plant-glycan-specific antibodies that could reduce therapeutic efficacy or cause adverse effects. By knocking out specific genes required for plant-specific glycosylation patterns and inserting mammalian glycosylation genes, glycoengineering generates plant hosts that produce mAbs with authentic human N-glycans10. Furthermore, plant-produced mAbs also have a degree of glycan homogeneity that cannot be produced by mammalian cells or by in vitro treatments11,12. This represents an advantage during the regulatory approval stage of product development. The availability of a portfolio of plant lines that can produce biologics with tailor-made mammalian N-glycans on demand provides the opportunity to develop vaccines and therapeutics with more potent efficacy or safety than those produced in other production platforms.\n\nPlant cells may also provide a novel vehicle for oral delivery of biologics. Conventional biologics are produced by a costly downstream process and require continuous refrigeration, referred to as the ‘cold-chain’, for their transport and storage plus sterile needles for injection. Oral delivery of protein drugs is appealing but has been elusive due to their denaturation and degradation in the digestive system and inability to cross the gut epithelium and subsequently deliver to target cells. Since human digestive enzymes cannot hydrolyze the glycosidic bonds in the carbohydrates of the plant cell wall, plant cells can protect expressed biologic proteins from acids and enzymes in the stomach by bioencapsulation, allowing them to enter the gut lumen where they are enzymatically released by gut commensal bacteria13. Recent studies also demonstrated that orally delivered plant cell-encapsulated protein drugs can cross the gut epithelium to enter the bloodstream. Depending on the specific targeting sequences they are fused to, the orally delivered protein drugs can either induce tolerance against inhibitory antibody production associated with their injection or enter the circulatory system to treat diabetes, hypertension, or other metabolic diseases13–15. Encapsulated protein drugs in plant cells have been found to maintain their pharmacological efficacy several years after storage at room temperature16.\n\nThese striking results suggest that plant-cell-encapsulated biologics may represent an ambient temperature-stable product that can be ingested by patients. These temperature-stable products allow the practical implementation of healthcare programs in regions where the ‘cold-chain’ and other logistical challenges limit the delivery of medical supplies. The commercial implementation of this strategy in the developed world would also reduce the cost associated with downstream processing, cold storage, and transportation. However, the regulatory challenges of this technology must be addressed, as vaccines or therapeutic drugs are required to have strictly controlled dosages, which is currently difficult to achieve with cell-encapsulation technology. Nevertheless, as new expression vectors provide more consistent biologic accumulation per unit of plant mass, this strategy may eventually offer an attractive future option for biologic delivery in both the developed and the developing world.\n\n\nSuccesses and remaining challenges\n\nThe story of ZMapp™ highlights how innovations in several basic technologies can come together and lead to the development of a life-saving drug candidate2. The development of magnICON vectors allowed for the rapid and high-level accumulation of anti-Ebola GP1 mAbs in Nicotiana benthamiana plants17,18. Host optimization permitted the production of these mAbs with various mammalian glycoforms, leading to the discovery that plant-derived anti-Ebola mAbs with homogenous GnGn mammalian glycans have a superior potency to their mammalian-produced counterparts12,19. Progress in downstream processing of plant materials allowed for the effective extraction and purification of these mAbs20,21. Efficacy studies in rhesus macaques demonstrated that a three-mAb cocktail was able to rescue 100% of animals even when given 5 days after a lethal Ebola challenge22. All of these paved the way for the formulation of ZMapp™ and its compassionate use in Brantly, Writebol, and five other human patients during the Ebola outbreak. A clinical trial has recently been concluded in the U.S., Liberia, Sierra Leone, and Guinea for ZMapp™ by the U.S. National Institutes of Health to assess the safety and efficacy of ZMapp™.\n\nAnother promising application of plant-based expression of biologics is the development of influenza virus-like particle (VLP)-based vaccines. Studies in this area have demonstrated the superiority of plant systems over other manufacturing platforms in their simplicity and speed for controlling potential pandemics23. An effective pandemic influenza vaccine needs to be produced in the shortest achievable timeframe to halt the spread of the new strain. VLPs comprising hemagglutinin (HA) alone are the simplest candidates for influenza pandemic vaccines because they require only the HA coding sequence of the pandemic strain for expression, impose fewer constraints on process and product characterization, and lower the risk of failure when production processes need to be adapted for a new viral strain23. However, producing VLPs based on HA alone is not feasible in animal cells because HA binds to the sialylated glycoproteins on the cell24. Plant cells provide a unique advantage for producing this VLP type because plant glycoproteins are not sialylated. The use of plants also avoids the supply issue of eggs in the event of massive culling of chickens, or if the influenza virus is lethal to embryonated eggs. Most importantly, the need for strain adaptation is eliminated when using plants, shortening the time required for vaccine production. In a real-life test in response to an unexpected outbreak of a novel A/H1N1 influenza virus, it took only 2 weeks to obtain infiltrated plants that expressed high levels of HA of the new strain and another 5 days to obtain the first purified lot of the vaccine from the date that the HA sequence of this strain became available23. This is in stark contrast with all current manufacturing technologies, which rely on strain adaptation, a process that requires an additional 4–6 months before vaccine production can be initiated. Efficacy studies in mice indicate the plant-derived VLPs have equivalent if not superior potency compared to vaccines produced in eggs23. The plant-derived vaccine candidates against various influenza strains (e.g. H5N and H1N1) have been tested in phase I and phase II human clinical trials. They were found to be safe and well tolerated, and the potency was among the most effective of the industry (www.medicago.com). This indicates that plant-based platforms provide an ideal system to produce biologics in response to emerging or re-emerging pathogens with unpredictable and frequent genetic drift or stockpile for bioterrorism threats.\n\nThe advantages of plant-based systems have been further demonstrated in the case of glucocerebrosidase (GCD), a therapeutic enzyme for treating Gaucher’s disease. Mammalian cell-produced GCD requires in vitro N-glycan processing to achieve the desired efficacy, substantially complicating the manufacturing process and increasing the production cost. In contrast, the plant-produced GCD already contains the required glycoform, eliminating the costly N-glycan processing and possibly resulting in better and more consistent efficacy25. As a result, the U.S. Food and Drug Administration (FDA) and other regulatory agencies have approved the use of plant-produced GCD (commercially named ELELYSO™) to treat Gaucher’s patients26. This is the very first PMB therapeutic ever approved by the FDA and it has been marketed in the U.S., Canada, and many Latin-American countries. A new oral-delivered version of GCD has been tested in animal models27 and in a phase I and a phase II human clinical trial (www.protalix.com). Results showed that the levels of GCD in the blood circulation of Gaucher’s patients were similar to those of healthy individuals when a juice containing lyophilized, GCD-containing carrot cells was consumed daily. The needle-free delivery of GCD will improve patients’ quality of life, encourage treatment compliance, and allow the implementation of therapy in areas where medical supplies are limited.\n\nThe application of PMBs has reached beyond the traditional realm of vaccines and therapeutic proteins. For example, plant-made bacterial colicins were recently shown to be very effective as food additives for controlling pathogenic bacteria in food products28. Even applied at low concentrations, the plant-derived colicins were shown to be highly and broadly active against all major pathogenic Escherichia coli strains that cause food poisoning. The production cost was estimated to be $1.00 per gram of purified colicins, indicating its commercial viability. The FDA’s “no questions” response letter to the commercialization request of plant-made colicins signals its potential regulatory approval. The commercialization of this product may significantly reduce bacterial enteric infections worldwide, as, currently, no effective methods are available to control pathogenic bacteria in the food chain.\n\nThere are still technical and regulatory challenges that must be overcome to fulfill the potential of PMBs. For example, the current manufacturing capacity of PMBs is still limited. This limitation was revealed when the demand for ZMapp™ during the 2014–2016 Ebola outbreak couldn’t be met even though a commercial-scale PMB facility devoted its full capacity to producing ZMapp™. Despite the successes of large-scale production of several PMBs, large-scale downstream processing of PMBs from whole plants remains challenging. Plants typically produce more solid debris than other organisms, and some plant species, such as those in the tobacco family, contain high levels of phenolics and alkaloids. As a result, clarification of plant extracts often cannot be achieved simply by a single round of filtration as is the case for mammalian cells, and direct loading of plant extracts onto chromatography resins may cause resin fouling20. As such, several rounds of filtration with multiple types of filters are required to remove plant particulates and/or plant compounds29. While these technologies can be scaled up to a certain extent, further optimization or the introduction of new processing methods is required since the full potential of PMB production on an agricultural scale demands a processing platform with extraordinarily large-scale capabilities.\n\nSince the launch of the PMB field in the early 1990s, there has been significant skepticism that a PMB product was ever going to be developed; this has now been done. The lingering criticism of the PMB field is the lack of approved human products in major biologic categories after more than 25 years of active research and development. To date, ELELYSO™ is the only PMB that has been approved by the FDA, and outside the compassionate use of ZMapp™ in human patients, no plant-made mAbs or vaccines have yet been licensed as pharmaceutical products for human use, albeit a plant-made mAb (CaroRx) that prevents adhesion of decay-causing bacteria to the tooth surface was approved as a medical device. The previous lack of a clear approval pathway is partially responsible, as the novelty and complexity of this technology caused uncertainty and confusion on how PMBs would fit into the regulatory agencies’ structured framework for biologics. The approval of ELELYSO™ by the FDA has paved a clear regulatory pathway specific for PMBs, especially for those derived from cultured plant cells, and should also streamline the approval of several whole-plant-made mAbs and vaccines that have shown safety and efficacy in human clinical trials30. The past uncertain regulatory environment also contributed to the decision by large pharmaceutical companies to forego PMBs. The recent progress made in the PMB field has, however, slowly warmed up their interests in the PMBs themselves or their production technologies. For example, Pfizer entered into an agreement to license the worldwide rights for commercializing ELELYSO™. Other pharmaceutical companies have also begun to show interest in PMBs through buyouts and partnerships31. The encouraging story of ZMapp™ has also sparked new interests and promoted several large government investments to expand the capacity of producing biologics from plants under current Good Manufacturing Practice (cGMP) regulations30,32.\n\n\nConclusions\n\nPlant production systems not only offer the traditional advantages of proper eukaryotic protein modification, low costs, high scalability, and increased safety but also allow the production of biologics at unprecedented speed to control potential pandemics or with specific post-translational modifications for superior potency or safety. These advantages make plants a superior alternative production system for biologic production. However, it is unlikely that plants will replace mammalian cells as the primary host for biologic production in the foreseeable future. Instead, plant-based systems will likely have a broad range of special niches for the production of specific biologics. Plants would be a system of choice to produce biosimilars because of their large capacity to rapidly generate biologics at low cost, the ability to easily incorporate post-translational modifications, and their low contamination risks with animal or human pathogens. For similar reasons, plants also offer an optimal system to produce biologics requiring extraordinarily large-quantity production that have relatively low profit margins. Plants will also be essential for producing safer and more effective biobetters due to the flexibility of producing biologics with specific and homogeneous mammalian glycoforms that cannot currently be easily produced by other cell culture systems. For the ultimate adoption and success of PMB technologies, it is crucial to overcome the scalability issue in downstream processing and vastly expand the approval pipeline of plant-made proteins in several key classes of biologics, such as mAbs and vaccines, within the next decade. Overall, the favorable outcome with ZMapp™ and the involvement of big pharma are promising signs and a harbinger of new optimism for the PMB field.\n\n\nAbbreviations\n\nPMBs, plant-made biologics; mAb, monoclonal antibody; VLP, virus-like particle; HA, hemagglutinin; GCD, glucocerebrosidase; FDA, US Food and Drug Administration; cGMP, good manufacturing practice.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no disclosures.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors thank J. Caspermeyer for the critical reading of the manuscript.\n\n\nReferences\n\nLyon GM, Mehta AK, Varkey JB, et al.: Clinical care of two patients with Ebola virus disease in the United States. N Engl J Med. 2014; 371(25): 2402–9. PubMed Abstract | Publisher Full Text\n\nArntzen C: Plant-made pharmaceuticals: from 'Edible Vaccines' to Ebola therapeutics. Plant Biotechnol J. 2015; 13(8): 1013–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nTusé D, Tu T, McDonald KA: Manufacturing economics of plant-made biologics: case studies in therapeutic and industrial enzymes. Biomed Res Int. 2014; 2014: 256135. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nChen Q, He J, Phoolcharoen W, et al.: Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Hum Vaccin. 2011; 7(3): 331–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKlimyuk V, Pogue G, Herz S, et al.: Production of recombinant antigens and antibodies in Nicotiana benthamiana using 'magnifection' technology: GMP-compliant facilities for small- and large-scale manufacturing. Curr Top Microbiol Immunol. 2014; 375: 127–54. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPeyret H, Lomonossoff GP: When plant virology met Agrobacterium: the rise of the deconstructed clones. Plant Biotechnol J. 2015; 13(8): 1121–35. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBendandi M, Marillonnet S, Kandzia R, et al.: Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin's lymphoma. Ann Oncol. 2010; 21(12): 2420–7. PubMed Abstract | Publisher Full Text\n\nWerner S, Breus O, Symonenko Y, et al.: High-level recombinant protein expression in transgenic plants by using a double-inducible viral vector. Proc Natl Acad Sci U S A. 2011; 108(34): 14061–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDugdale B, Mortimer CL, Kato M, et al.: In plant activation: an inducible, hyperexpression platform for recombinant protein production in plants. Plant Cell. 2013; 25(7): 2429–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStrasser R, Altmann F, Steinkellner H: Controlled glycosylation of plant-produced recombinant proteins. Curr Opin Biotechnol. 2014; 30: 95–100. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLai H, He J, Hurtado J, et al.: Structural and functional characterization of an anti-West Nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants. Plant Biotechnol J. 2014; 12(8): 1098–107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOlinger GG Jr, Pettitt J, Kim D, et al.: Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proc Natl Acad Sci U S A. 2012; 109(44): 18030–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKwon KC, Daniell H: Low-cost oral delivery of protein drugs bioencapsulated in plant cells. Plant Biotechnol J. 2015; 13(8): 1017–22. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSu J, Sherman A, Doerfler PA, et al.: Oral delivery of Acid Alpha Glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of Pompe mice. Plant Biotechnol J. 2015; 13(8): 1023–32. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSu J, Zhu L, Sherman A, et al.: Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B. Biomaterials. 2015; 70: 84–93. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLakshmi PS, Verma D, Yang X, et al.: Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro. PLoS One. 2013; 8(1): e54708. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang Z, Phoolcharoen W, Lai H, et al.: High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system. Biotechnol Bioeng. 2010; 106(1): 9–17. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCastilho A, Bohorova N, Grass J, et al.: Rapid high yield production of different glycoforms of Ebola virus monoclonal antibody. PLoS One. 2011; 6(10): e26040. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZeitlin L, Pettitt J, Scully C, et al.: Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant. Proc Natl Acad Sci U S A. 2011; 108(51): 20690–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuyel JF, Twyman RM, Fischer R: Extraction and downstream processing of plant-derived recombinant proteins. Biotechnol Adv. 2015; 33(6 Pt 1): 902–13. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFulton A, Lai H, Chen Q, et al.: Purification of monoclonal antibody against Ebola GP1 protein expressed in Nicotiana benthamiana. J Chromatogr A. 2015; 1389: 128–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQiu X, Wong G, Audet J, et al.: Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature. 2014; 514(7520): 47–53. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nD'Aoust MA, Couture MM, Charland N, et al.: The production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza. Plant Biotechnol J. 2010; 8(5): 607–19. PubMed Abstract | Publisher Full Text\n\nChen BJ, Leser GP, Morita E, et al.: Influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles. J Virol. 2007; 81(13): 7111–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZimran A, Brill-Almon E, Chertkoff R, et al.: Pivotal trial with plant cell-expressed recombinant glucocerebrosidase, taliglucerase alfa, a novel enzyme replacement therapy for Gaucher disease. Blood. 2011; 118(22): 5767–73. PubMed Abstract | Publisher Full Text\n\nFox JL: First plant-made biologic approved. Nat Biotechnol. 2012; 30: 472. Publisher Full Text\n\nShaaltiel Y, Gingis-Velitski S, Tzaban S, et al.: Plant-based oral delivery of β-glucocerebrosidase as an enzyme replacement therapy for Gaucher's disease. Plant Biotechnol J. 2015; 13(8): 1033–40. PubMed Abstract | Publisher Full Text\n\nSchulz S, Stephan A, Hahn S, et al.: Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins. Proc Natl Acad Sci U S A. 2015; 112(40): E5454–60. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBuyel JF, Fischer R: Downstream processing of biopharmaceutical proteins produced in plants: the pros and cons of flocculants. Bioengineered. 2014; 5(2): 138–42. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMa JK, Drossard J, Lewis D, et al.: Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants. Plant Biotechnol J. 2015; 13(8): 1106–20. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPaul MJ, Thangaraj H, Ma JK: Commercialization of new biotechnology: a systematic review of 16 commercial case studies in a novel manufacturing sector. Plant Biotechnol J. 2015; 13(8): 1209–20. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHoltz BR, Berquist BR, Bennett LD, et al.: Commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals. Plant Biotechnol J. 2015; 13(8): 1180–90. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "13893",
"date": "19 May 2016",
"name": "Kenneth Palmer",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13898",
"date": "19 May 2016",
"name": "Larry Zeitlin",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13899",
"date": "19 May 2016",
"name": "Rainer Fischer",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-912
|
https://f1000research.com/articles/5-911/v1
|
19 May 16
|
{
"type": "Opinion Article",
"title": "Is erythropoietin a worthy candidate for traumatic brain injury or are we heading the wrong way?",
"authors": [
"Giovanni Grasso",
"Concetta Alafaci",
"Pietro Ghezzi",
"Concetta Alafaci",
"Pietro Ghezzi"
],
"abstract": "Traumatic brain injury (TBI) is a leading cause of death and disability in the modern society. Although primary prevention is the only strategy that can counteract the primary brain damage, numerous preclinical studies have been accumulated in order to find therapeutic strategies against the secondary damage. In this scenario erythropoietin (EPO) has been shown to be a promising candidate as neuroprotective agent. A recent clinical trial, however, has shown that EPO has not an overall effect on outcomes following TBI thus renewing old concerns. However, the results of a prespecified sensitivity analysis indicate that the effect of EPO on mortality remains still unclear. In the light of these observations, further investigations are needed to resolve doubts on EPO effectiveness in order to provide a more solid base for tailoring conclusive clinical trials.",
"keywords": [
"Traumatic brain injury",
"Neuroprotection",
"Erythropoietin"
],
"content": "\n\nTraumatic brain injury (TBI) is one of the major causes of death and disability in our society1. TBI can provides heterogeneous effects since, in addition to the primary injury, it is associated with the so-called secondary brain injury where inflammation, excitotoxicity, ischemia, edema participate in worsening the clinical scenario13. Several pre-clinical studies have been conducted in order to identify neuroprotective agents able to counteract the secondary tissue damage and improve clinical outcomes8. However, translation to the clinical trials has been discouraging and the treatment of TBI remains great challenge worldwide.\n\nIn both pre-clinical and clinical studies, erythropoietin (EPO) has been recognized for nearly two decades as a potent neuroprotective agent with a multifaceted, hematopoiesis-independent action profile4. The discovery that EPO has neuroprotective functions apart from regulating erythropoiesis3 was unexpected and prompted numerous studies showing a protecting role through antiapoptotic, antioxidative and anti-inflammatory, angiogenic and neurotrophic mechanisms7,9.\n\nThe recent conclusion of the EPO-TBI, double-blind randomized controlled trial10, has renewed old concerns. This clinical study was undertaken in 29 centers in seven countries. A total of 606 patients were randomly selected. EPO was given to 308 patients in a dose of 40,000 units subcutaneously, while 298 patients received a placebo, consisting of 0.9% sodium chloride,. Both EPO and placebo were administered once per week for a maximum of three doses. Randomization was stratified by severity of traumatic brain injury (moderate vs. severe) and participating site10. The primary outcome, consisting of improvement in the patients’ neurological status was measured at 6 months follow-up. It was summarized as a binary midpoint reduction of their extended Glasgow Outcome Scale (GOS-E) level, which was defined as a GOS-E of 1–4 (death, vegetative state, and severe disability) or a GOS-E of 5–8 (moderate disability and good recovery). In addition, mortality, proximal deep venous thrombosis and occurrence of general thrombotic events were assessed as secondary outcomes measures10.\n\nThe authors found that EPO did not reduce the number of patients with a GOS-E level of 4 or lower, and did not affect the incidence of deep venous thrombosis events.\n\nOverall, the results of this international multicenter randomized placebo-controlled trial suggest that EPO may not be useful in TBI. This result is in contrast with a number of experimental studies suggesting that EPO might improve neurological outcomes following TBI. However, the results of a prespecified sensitivity analysis adjusting for covariates indicate that the effect of EPO on mortality remains to be better investigated. Notably, although in this study EPO did not have an overall effect on survival10, when adjusted for illness severity according to the IMPACT-TBI predicted probability of a poor outcome, 6-months mortality was lower in patients given EPO than in those who received placebo.\n\nAlthough the authors suggest caution in the interpretation of these mortality findings, we believe this question is worthy of note and remains to be addressed. The time window for EPO administration following TBI and its dose regimen are the main arguments. In this study a 24 hours time window and a dose of 40,000 units was chosen. It must be taken into account that earlier preclinical studies showed that recombinant human EPO treatment at a dose of 1000 IU/kg administered every 8 hours starting following TBI, is effective as neuroprotective agent5. The dose used in the study by Nichol and collaborators14 is the lowest dosage known to be effective in the experimental settings, and the time for the first administration, an average of 18.6 hours after TBI, would initiate a neuroprotective program in a late secondary damage. The small dose used, time and frequency of administration could contribute to the unfavorable results from this clinical trial. Neuroprotective drugs should be administered as soon as possible and as long as the pathological cascades occur. EPO dose and therapeutic duration were clearly dictated by the concerns on the safety of recombinant human EPO. It is well known that all the information available regarding the safety of EPO comes from its non-neurologic use15. Using the information accumulated on EPO safety in patients affected by chronic anemia and put into practice for the management of TBI can be dangerous since the interaction between EPO and various physiologic variables, in addition to drugs commonly used in TBI patients, are unknown.\n\nAdditionally, besides its fame of a well-tolerated drug, recent reports of adverse effects associated with the chronic administration of recombinant EPO (i.e. hypertension, hypertensive encephalopathy, seizures, and thrombotic/vascular events) have raised new concerns6. Although in experimental and clinical studies, including this randomized trial, no adverse effects during EPO treatment were observed, it is unknown what the effect in patients with a raised hemoglobin concentration would be.\n\nTaken collectively, the findings of this recent clinical trial11, together with those from previous randomized studies2,12,14, suggest that EPO might decrease mortality in this patient group.\n\nThe overall disappointing results of the clinical trials reported over the time could be due to protocol and dosage problems, and one should also bear in mind that it is not always possible to translate animal research to the clinical scenario, which is more complex and less controlled.\n\nMore attention should be paid in conducting clinical trials in order to obtain sufficient information regarding therapeutic time window, dosage, duration of therapy and safety. The uncertain results so far obtained put EPO at risk of being discarded as thoroughly as it was initially welcomed as a miracle drug. Meanwhile, better information on the spectrum of biological actions of EPO and the underlying mechanisms would provide a more solid base for tailoring conclusive clinical trials.\n\nIn the light of these observations, further investigations are required to resolve such uncertainties especially when issues as optimal dosages, therapeutic time window, and duration of therapy deserve to be clarified.",
"appendix": "Author contributions\n\n\n\nGG prepared the manuscript, CA contributed to the preparation of the manuscript and revision, PG offered data interpretation.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nPG has received research funds and travel money from Araim Pharma and Warren Pharma, who are developing EPO derivatives with neuroprotective properties.\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\nCenters for Disease Control and Prevention (CDC): Rates of hospitalization related to traumatic brain injury--nine states, 2003. MMWR Morb Mortal Wkly Rep. 2007; 56(8): 167–170. PubMed Abstract\n\nAbrishamkar S, Safavi M, Honarmand A: Effect of erythropoietin on Glasgow Coma Scale and Glasgow Outcome Sale in patient with diffuse axonal injury. J Res Med Sci. 2012; 17(1): 51–56. PubMed Abstract | Free Full Text\n\nBrines ML, Ghezzi P, Keenan S, et al.: Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Natl Acad Sci U S A. 2000; 97(19): 10526–10531. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrasso G, Sfacteria A: The many faces of erythropoietin: from erythropoiesis to a rational neuroprotective strategy. Expert Opin Investig Drugs. 2008; 17(10): 1407–1409. PubMed Abstract | Publisher Full Text\n\nGrasso G, Sfacteria A, Meli F, et al.: Neuroprotection by erythropoietin administration after experimental traumatic brain injury. Brain Res. 2007; 1182: 99–105. PubMed Abstract | Publisher Full Text\n\nJia L, Chopp M, Zhang L, et al.: Erythropoietin in combination of tissue plasminogen activator exacerbates brain hemorrhage when treatment is initiated 6 hours after stroke. Stroke. 2010; 41(9): 2071–2076. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJuul SE, Beyer RP, Bammler TK, et al.: Microarray analysis of high-dose recombinant erythropoietin treatment of unilateral brain injury in neonatal mouse hippocampus. Pediatr Res. 2009; 65(5): 485–492. PubMed Abstract\n\nKabadi SV, Faden AI: Neuroprotective strategies for traumatic brain injury: improving clinical translation. Int J Mol Sci. 2014; 15(1): 1216–1236. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaiese K, Chong ZZ, Hou J, et al.: Erythropoietin and oxidative stress. Curr Neurovasc Res. 2008; 5(2): 125–142. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNichol A, French C, Little L, et al.: Erythropoietin in traumatic brain injury (EPO-TBI): a double-blind randomised controlled trial. Lancet. 2015; 386(10012): 2499–506. PubMed Abstract | Publisher Full Text\n\nNichol A, French C, Little L, et al.: Erythropoietin in traumatic brain injury: study protocol for a randomised controlled trial. Trials. 2015; 16: 39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNirula R, Diaz-Arrastia R, Brasel K, et al.: Safety and efficacy of erythropoietin in traumatic brain injury patients: a pilot randomized trial. Crit Care Res Pract. 2010; 2010: pii: 209848. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPark E, Bell JD, Baker AJ: Traumatic brain injury: can the consequences be stopped? CMAJ. 2008; 178(9): 1163–1170. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobertson CS, Hannay HJ, Yamal JM, et al.: Effect of erythropoietin and transfusion threshold on neurological recovery after traumatic brain injury: a randomized clinical trial. JAMA. 2014; 312(1): 36–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTong EM, Nissenson AR: Erythropoietin and anemia. Semin Nephrol. 2001; 21(2): 190–203. PubMed Abstract"
}
|
[
{
"id": "13929",
"date": "23 May 2016",
"name": "Hannelore Ehrenreich",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEPO, TBI and some more to say\nIn their opinion article, “Is erythropoietin a worthy candidate for traumatic brain injury or are we heading the wrong way?” Giovanni Grasso, Concetta Alafaci and Pietro Ghezzi address a very important topic. Specifically, they summarize and comment on recent formally negative clinical trials on the use of recombinant human erythropoietin (EPO) in traumatic brain injury (TBI). More globally, they lay their fingers in the wound of numerous clinical trials on neuroprotection and neuroregeneration in brain diseases that failed in translation from preclinical studies to the patient. The authors discuss briefly properties and encouraging preclinical studies on EPO, and then raise the most critical issues of the human EPO trials in TBI, mainly the late administration of the first dose, the short duration and low frequency of treatment - all essentially dictated by not too well established safety concerns.\nWhile we fully agree on most of these points, we do not think that the single dose was necessarily too low, as also criticized by the authors. We feel that the dose was likely sufficient but that intravenous rather than subcutaneous dosing should have been used to achieve higher EPO levels in the brain.\nWe would like to add a few comments that we feel – after 20 years of own experience in translational work on the brain EPO system – are important to consider.\nWe had to painfully learn ourselves the downstream consequences of pharmaceutical companies and overeager regulatories drawing fast conclusions out of too superficially or not at all analyzed data. This triggered an avalanche of destruction regarding our large EPO stroke multicenter trial where severe treatment violations of stroke patients in several centers (totally independent of the study medication) explained the outcome rather than EPO1. In fact, careful subpopulation analysis of all dead patients revealed that several relevant baseline characteristics (i.e. data obtained before administration of any study medication) were significantly different between groups, always in disadvantage of the EPO group. Most importantly, upon inclusion (before any study drug application), intent-to-treat non-rtPA receiving EPO patients who died suffered from much severer strokes as compared to placebo patients (NIHSS day 1: 20.4±5.4 versus 13.3±4.9; p=0.003). This highly significant prediction of a worse outcome explains the twofold higher very early death rate in the EPO group (http://www.epo-study.de/index_eng.html).\nUnfortunately, the premature jumping to conclusions regarding the EPO stroke trial influenced also one of the TBI trials discussed here: The authors write that “there was concern by the FDA that the initial regimen of 3 daily doses of EPO would impose a greater risk of death. This concern resulted in a modified study design after approximately one-third of the patients had been enrolled in the trial. We did not detect an increased mortality rate with the EPO dose regimen, and the neurological outcome results were more promising than with the subsequent regimen. However, because the original dose regimen was stopped early, the numbers of cases are too small to draw any conclusions”2.\nEven though safety is undoubtedly an important issue, we have to reduce the incredible arrogance of dismissing any trial that shows ‘just signals’ of benefit. How can we expect more in an initial translational step? Being aware that there are no neuroprotective/neuroregenerative treatments out for conditions as frequent and detrimental as stroke or TBI, it would be time to work hard on any positive signals rather than ‘throw the baby out with the bathwater’.\nAnd clearly, both TBI trials2,3 show such signals. Regarding stroke, not only the first EPO trial was obviously promising4, but also the retrospective analysis of patients from Hannover, the most efficiently recruiting center of the second EPO stroke trial5, made the beneficial effect of EPO in stroke again very obvious.\nGetting back to the opinion paper by Grasso and colleagues, longer treatment duration - over many weeks - may ultimately disclose the benefit of EPO also for TBI much stronger. Clinical studies on EPO in chronic brain diseases (schizophrenia, multiple sclerosis, major and bipolar depression) with extended treatment using high dose EPO over many weeks showed consistently advantageous effects on cognition, motor function, and even reduction of brain matter loss. All these findings were in absence of any appreciable side effects6-11. Of course, in all clinical studies, the quality of patient care including alert follow-up of individual patients at all times is mandatory12.\nWork on EPO indications outside the hematopoietic system has been difficult ever since. Large studies would be needed with adequate funding. Funding agencies do not provide enough money and direct requests for financing to industry. Industry has not been supportive due to expired EPO patents and many EPO biosimilar producers popping up, increasing the risk of ‘off-label-use’. Protection of the extremely lucrative anemia market includes avoiding the risk of additional side effects in new indications.\n\nEPO is a potent growth factor, not a miracle drug, and it is not a causal treatment or cure of brain diseases but it may improve their outcome. Even though doping is an unpleasant chapter in itself, it may be seen in the present context as one of the most convincing field studies ever regarding efficiency of EPO. Who would invest huge amounts of money over decades and risk to be caught by controllers and convicted if EPO were not extremely effective?\nDespite all frustrations and disappointments: We have to keep trying to understand how EPO acts in the brain and to ultimately exploit this knowledge for the benefit of our patients. Some novel and surprising insight, lending further support to the use of EPO for neuroprotective and neuroregenerative treatment of brain diseases, comes from recent studies showing that EPO increases the number of neurons and oligodendrocytes in the hippocampus by driving pre-existing precursors to differentiate13, or from work reporting EPO effects on synaptic plasticity14,15.",
"responses": []
},
{
"id": "13936",
"date": "24 May 2016",
"name": "Alessandro Landi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 exhaustive and clear, appropriate for the content. The study design, methods and analysis are well conduct and appropriate for the topic. The conclusions are relevant in this field.\n\nI think that this article is suitable in this form",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-911
|
https://f1000research.com/articles/5-907/v1
|
18 May 16
|
{
"type": "Review",
"title": "Upper extremity nerve block: how can benefit, duration, and safety be improved? An update",
"authors": [
"Metha Brattwall",
"Pether Jildenstål",
"Margareta Warrén Stomberg",
"Jan G. Jakobsson",
"Metha Brattwall",
"Pether Jildenstål",
"Margareta Warrén Stomberg"
],
"abstract": "Upper extremity blocks are useful as both sole anaesthesia and/or a supplement to general anaesthesia and they further provide effective postoperative analgesia, reducing the need for opioid analgesics. There is without doubt a renewed interest among anaesthesiologists in the interscalene, supraclavicular, infraclavicular, and axillary plexus blocks with the increasing use of ultrasound guidance. The ultrasound-guided technique visualising the needle tip and solution injected reduces the risk of side effects, accidental intravascular injection, and possibly also trauma to surrounding tissues. The ultrasound technique has also reduced the volume needed in order to gain effective block. Still, single-shot plexus block, although it produces effective anaesthesia, has a limited duration of postoperative analgesia and a number of adjuncts have been tested in order to prolong analgesia duration. The addition of steroids, midazolam, clonidine, dexmedetomidine, and buprenorphine has been studied, all being off-label when administered by perineural injection, and the potential neurotoxicity needs further study. The use of perineural catheters is an effective option to improve and prolong the postoperative analgesic effect. Upper extremity plexus blocks have an obvious place as a sole anaesthetic technique or as a powerful complement to general anaesthesia, reducing the need for analgesics and hypnotics intraoperatively, and provide effective early postoperative pain relief. Continuous perineural infusion is an effective option to prolong the effects and improve postoperative quality.",
"keywords": [
"upper extremity block",
"peripheral blocks",
"ultra-sound guided",
"anaesthesia",
"interscalene",
"supraclavicular",
"infraclavicular",
"axillary plexus"
],
"content": "Introduction\n\nPeripheral blocks have been part of anaesthetic techniques used for upper extremity surgery for decades. Intravenous regional anaesthesia, the so-called Bier block, was described in 1908 by A. G. Bier1. The interscalene block was also initially described more than a century ago, in the early 1900s2. The brachial plexus came some years later3. These blocks were initially done by identifying anatomical landmarks and eliciting paraesthesia. The introduction of the nerve stimulator technique in the late 1980s made a marked change in practice: from being a technique used only by experienced individuals, it became far more commonly used4. The introduction of the ultrasound technique during the last decade has further enhanced performance. A Cochrane review suggests that the ultrasound-guided block technique further improves the success and ease of performance5. The availability of information via websites6 and demo films7,8 has made the technique much appreciated, and it is now commonly used by younger colleagues. A recent paper by Sehmbi et al. describes the ultrasound-guided technique for various upper extremity blocks9. Ultrasound guidance further increases the success rate and, when used in combination with nerve stimulation, it provides, as of today, the highest degree of safety and success10,11. A recent Cochrane systematic review supports the efficacy of the ultrasound-guided block technique but also addresses the importance of experience and the training curve of ultrasound vs. other techniques5. In the March-April 2016 issue of Regional Anesthesia and Pain Medicine, Neal et al.12 have also provided an executive summary of “Evidence-Based Medicine Assessment of Ultrasound-Guided Regional Anesthesia”. They concluded that there is high-level evidence supporting ultrasound guidance, contributing to superior characteristics with selected blocks, although absolute differences with the comparator technique, e.g. nerve stimulation, are often relatively small, especially for upper extremity blocks.\n\nThe upper extremity blocks may be divided into the following13:\n\nInterscalene, shoulder surgery\n\nSupraclavicular, the entire arm\n\nInfraclavicular, the elbow and below\n\nAxillar plexus, from below the elbow\n\nThe aim of the present paper is to provide an update and overview of the clinical usage of upper extremity nerve blocks, how analgesic effect can be prolonged, and how safety can possibly be improved, strengthening the benefit vs. risk for their clinical use.\n\nWe conducted a public domain literature search using PubMed looking for papers addressing upper extremity block, ultrasound-guided block, and pain, with a focus on meta-analyses and reviews published in 2010 and onwards.\n\n\nEfficacy and outcome\n\nToday, the interscalene block is well established for intraoperative as well as postoperative pain management associated with shoulder surgery. In May 2015, Abdallah et al. performed a meta-analysis14, which showed a clear effect on pain for up to 6 hours during movement and 8 hours at rest and an opioid-sparing effect for up to 24 hours after surgery. Ullah et al.15 searched for studies assessing continuous perineural interscalene block for pain relief after major shoulder surgery, but they were not able to conduct a meta-analysis because of the lack of studies. They still concluded that the catheter technique provides better pain relief than parenteral analgesics. There are further studies supporting the beneficial effects of the perineural catheter technique improving postoperative pain course. Fredrickson et al.16 compared single-shot to continuous perineural infusion following minor shoulder surgery and found significant positive effects. Salviz et al.17 likewise found superior postoperative analgesia up to day 7 when comparing the single-shot to the catheter technique. Patients in the continuous interscalene block group received 20 mL of 0.5% ropivacaine as a bolus through a catheter, whereas single-shot patients received the same injection volume through a needle. The continuous group of patients received a further infusion of 0.2% ropivacaine at 5 mL/hour with a patient-controlled bolus of 5 mL hourly for 48 hours. Mariano et al.18 compared single-shot to a 48-hour continuous infusion with ropivacaine 0.2 mg/mL. They also found superior pain relief, improved sleep, and higher patient satisfaction. Indeed, perineural catheter techniques are an effective option.\n\nSupraclavicular block carries the risk of pneumothorax and also the development of transient Horner’s syndrome. However, the ultrasound-guided technique has facilitated its performance, and there is a growing interest in the block. In 2014, Sadowski et al. published a comprehensive review on its renascence following the introduction of the ultrasound-guided technique19. Gamo et al. presented their experience with the ultrasound-guided supraclavicular block technique in 202 patients20. They showed the block had a rapid procedure time (average 4 minutes), good intraoperative conditions, a mean surgery time of 75.2 minutes with a range of 6 to 232, and a mean of 437 minutes (range 171 to 992) of postoperative analgesia. Transient Horner’s syndrome was observed in 10% of patients. Vaghadia et al. compared ropivacaine and bupivacaine for supraclavicular plexus block performed by paraesthesia or nerve stimulation technique in 104 ASA physical status 1–3 patients scheduled for upper arm surgery. Long and effective anaesthesia/analgesia was achieved with ropivacaine 7.5 mg/mL similar to 5 mg/mL bupivacaine without differences between the two groups. The mean duration of analgesia, time to need for rescue analgesia, was 11–12 hours21.\n\nThere is one recent meta-analysis assessing the available evidence on the infraclavicular block technique for perioperative use. In 2013, Chin et al. published a Cochrane systematic review on the use of infraclavicular plexus block for surgery of the lower part of the arm22. They concluded, based on the 15 studies included, that infraclavicular plexus block is an effective alternative to supraclavicular and axillary block, providing superior intraoperative tourniquet pain control as compared to single injection axillary block and faster performance as compared to multi-injection axillary block. It had a similar postoperative analgesic duration as compared to other peripheral blocks (supraclavicular and axillary). Overall, it seems to be an advantageous technique over the traditional axillary block.\n\nChin et al. also conducted a Cochrane systematic review on axillary plexus block, assessing single, double, and multiple injection techniques23. In all, 21 trials were included, presenting results from a total of 2148 participants who received regional anaesthesia for hand, wrist, forearm, or elbow surgery. Studies with trans-arterial and nerve stimulator techniques were included. The multiple injection technique was found to improve success rates but demonstrated adequate surgical anaesthesia and motor block as compared to the single injection technique. No significant difference was found in analgesia failure, complications, and patient discomfort. However, the time for block performance was significantly shorter for single and double injection techniques as compared with multiple injections. There are two recent papers comparing nerve stimulation and ultrasound guidance for axillary plexus block. Kumar et al. found both techniques to be equally safe and effective24. Meierhofer et al. found similar results25. No major difference in success rate between nerve stimulation and ultrasound technique was found; however, the authors commented that the skills required for each respective technique must be taken into account.\n\n\nRisk and side effects\n\nThe upper extremity block may cause side effects, such as nerve damage, intravascular injection causing local anaesthesia toxicity, diaphragm dysfunction, and pneumothorax. There is a recent update from the American Society of Regional Anesthesia and Pain Medicine by Neal26: this analysis concluded that ultrasound guidance has no significant effect on the incidence of postoperative neurologic symptoms. The ultrasound-guided block technique reduces the incidence and intensity of hemidiaphragmatic paresis but in an unpredictable manner. Ultrasound guidance reduces the risk of local anaesthesia toxicity and may also reduce the predicted frequency of pneumothorax, but this requires training in visualisation of the needle27. Also, with regard to the volume and concentration, the lower dosage needed has an impact with less of an effect on the diaphragm28,29. Thackeray et al.30 showed that an ultrasound-guided interscalene brachial plexus catheter placement with 20 mL of 0.125% bupivacaine caused significantly less diaphragm dysfunction as compared to 0.25% bupivacaine. Stundner et al.31 conducted an elegant study, administering patients with ropivacaine 0.75%, either 20 or 5 mL, plus contrast dye followed by magnetic resonance imaging. Both groups experienced fast onset and adequate intraoperative and postoperative analgesia, with no significant differences in pain scores. The spread was more pronounced with the higher volume, diaphragm dysfunction occurred twice as frequently, and changes from baseline peak respiratory flow rate were in the 20 mL group. Horner’s syndrome may also be associated with upper extremity plexus blocks. Tran et al.32 compared ultrasound-guided supraclavicular, infraclavicular, and axillary brachial plexus blocks for upper extremity surgery of the elbow, forearm, wrist, and hand. They found all three blocks to be effective; however, the axillary block required longer time to perform and the supraclavicular block was associated with a higher incidence of Horner’s syndrome (37.5% vs. 0−5%; both P <0.001).\n\n\nTechniques in order to facilitate block quality and duration\n\nThe single-shot upper extremity blocks have effective anaesthesia duration of hours when performed with a long-lasting local anaesthetic (bupivacaine, levobupivacaine, or ropivacaine). The analgesic effect wears off within the duration for a long-lasting local anaesthetic (6–10 hours). Abdallah et al.14 raised the question of whether the benefit of the early pain relief may be overwhelmed by the pain “rebound” when the block wears off. Pain during the first postoperative evening and night following discharge does cause concern. Sunderland et al. found that patients who had a block required more unplanned healthcare visits as compared to patients who had general anaesthesia, mostly due to pain33. There is without doubt an interest in prolonging the analgesic effect, reducing postoperative pain, and this can be done by the addition/combination of different adjuncts. There are several techniques used in order to improve quality and extend the duration of postoperative analgesia, and different adjuncts have been tested in order to facilitate the quality and the duration of peripheral nerve blocks. It must, however, be acknowledged that the use of adjuncts for perineural administration is off-label and potential nerve toxicity needs further study. Adding active medication perineurally must be done with the benefit and potential risk taken into account for each individual patient. Williams et al.34 conducted an in vitro study assessing the neurotoxicity from ropivacaine sole agent and in combination with clonidine, buprenorphine, dexamethasone, and midazolam and showed clear signs of neurotoxicity, suggesting that further studies are warranted in order to elucidate risk. Kirksey et al. published a systematic review in September 2015 on adjuncts for improving peripheral blocks, and they concluded that the addition of perineural buprenorphine, clonidine, dexamethasone, dexmedetomidine, or magnesium showed a consistently prolonged duration of peripheral nerve block35. The benefit vs. risk associated with the co-administration of drugs perineurally must, however, be acknowledged. Bailard et al. addressed the benefit vs. risk and commented that there are an increasing number of studies suggesting that systemic administration may provide more or less similar effects to perineural administration and possibly a decreased risk, at least for nerve toxicity36.\n\n\nClonidine\n\nAlpha-2-agonists have been added to upper extremity blocks for decades. In 1996, Singelyn et al. conducted a dose-finding study assessing the minimal effective clonidine dose able to improve axillary block37. Axillary brachial plexus block was performed with nerve stimulator and 40 mL 1% mepivacaine plus 1:200,000 epinephrine local anaesthesia doses. The control group received no clonidine. In the other groups, increasing doses of clonidine (0.1, 0.2, 0.3, 0.4, 0.5, 1, and 1.5 mg/kg) were added to the local anaesthetic solution. Both anaesthesia and analgesia duration were increased, with the lowest effective dose being 0.5 mg/kg clonidine, with no clear additional effect in further increasing the dose. The anaesthesia duration was increased from 260 minutes up to 310 at 0.5 mg/kg, where the effect plateaued, and the analgesia showed a small dose response from 260 minutes up to 490 minutes, with only limited additional effects from 1.0 and 1.5 mg/kg. The linear trend in the duration of analgesia corresponding to doses of 0.5, 1, and 1.5 pg/kg was not significant (P = 0.97), indicating no further increase in duration with dose. No effect on the onset or quality of block was noticed. McCartney et al. performed the first meta-analysis on the effects of adding clonidine to peripheral blocks38. In all, 1385 patients in 27 studies were included, and five studies included a systemic control group. The dose of clonidine studied ranged from 30 to 300 mg. There were 15 studies that supported the use of clonidine as an adjunct to peripheral nerve block, with 12 studies failing to show any benefit. Clonidine appeared to prolong analgesia when added to intermediate-acting local anaesthetics for axillary and peribulbar blocks. In 2009, Pöpping et al. conducted a meta-analysis of the available evidence on the addition of clonidine to peripheral blocks and concluded that there was still a lack of clear evidence of dose responsiveness for beneficial as well as for harmful effects39. The combination of clonidine and intermediate or long-acting local anaesthetics for single-shot peripheral nerve or plexus blocks prolongs the duration of analgesia and motor block by about 2 hours. They also commented on the potential risk of alpha-2-agonist-associated side effects; therefore, increased risk of hypotension, fainting, and sedation may limit its usefulness. Furthermore, the optimal dose, the dose responsiveness to balance benefit and duration, and the side effects remain unclear.\n\n\nDexmedetomidine\n\nSwami et al. conducted a randomised double blind study in 60 patients where the same dosage of clonidine or dexmedetomidine as an adjuvant to bupivacaine in supraclavicular brachial plexus block was compared40. There was no statistically significant difference in onset of sensory and motor block between the two groups. They found in this small study, however, that dexmedetomidine provided superior postoperative effects as compared to clonidine. The duration of sensory block and motor block was mean 227 ± 48 and 292 ± 59 minutes, respectively, in the clonidine group of patients and mean 414 ± 87 and 472 ± 90 minutes, respectively, in the dexmedetomidine group. The duration of analgesia (time to requirement of rescue analgesia) in the dexmedetomidine group was 456 ± 97 minutes, while in the clonidine group it was 289 ± 62 minutes. Statistically, this difference was significant (P=0.001). The number of patients achieving what was considered an excellent block was also higher in the dexmedetomidine group of patients (80%) as compared with the clonidine group (40%) (P <0.05). In 2013, Abdulla et al. conducted a meta-analysis of studies assessing the co-administration of dexmedetomidine to perineural blocks41. Four out of the nine studies included were studies of brachial plexus blocks. Dexmedetomidine significantly increased analgesia duration for spinal block, but the increase was not significant for the plexus block studies. The duration on motor function and time to first analgesic request were prolonged for both intrathecal and brachial plexus block. The authors concluded that there are still insufficient safety data to support perineural dexmedetomidine use in the clinical setting. Wu et al. performed a second meta-analysis, which was published in early 201442. In all, 1092 patients from 16 randomised controlled clinical trials were included in the analysis. They found, likewise, that neuraxial dexmedetomidine improved postoperative analgesia, with a mean difference of almost 7 hours, and significantly decreased postoperative pain intensity. Dexmedetomidine co-administration, however, increased the risk of bradycardia, with an odds ratio of 2.68.\n\nThe most recent study from September 2015 by Kaur et al.43 assessing the effects of dexmedetomidine for plexus block also found a statistical difference between the groups in motor blockade and postoperative pain with an advantage seen in the group of patients who received dexmedetomidine. They also noticed some minor haemodynamic side effects from dexmedetomidine, such as bradycardia in two patients; however, none of these patients needed any treatment.\n\n\nDexamethasone\n\nIn 2014, Choi et al. conducted a systematic review and meta-analysis of randomised trials comparing brachial plexus block performed with local anaesthetic with or without additional perineural dexamethasone44. In all, 801 patients from nine trials were included, with 393 patients receiving dexamethasone (4–10 mg). The meta-analysis showed that dexamethasone prolonged the analgesic duration for long-acting local anaesthetics from 730 to 1306 minutes, with a mean difference of 576 minutes, and for intermediate local anaesthetics from 168 to 343 minutes, with a mean difference of over 175 minutes. The motor block effect was also prolonged from 664 to 1102 minutes. An important finding was that the most recent trial demonstrated equivalent prolongation with perineural or systemic administration of dexamethasone compared with placebo. Also in 2014, de Oliveira Jr et al.45 published a review article regarding perineural dexamethasone as an adjunct to the brachial plexus blocks. In all, 760 subjects from nine randomised trials with dexamethasone were included. They found a clear positive effect of perineural dexamethasone over control for analgesia, a mean prolongation of 473 (264 to 682) minutes, and a motor blockade duration of 500 (154 to 846) minutes. Postoperative opioid consumption was also reduced in the perineural dexamethasone group compared to control (-8.5 [-12.3 to -4.6] mg of intravenous morphine equivalents). In 2015, Albrecht et al. published a systematic review and meta-analysis regarding the safety and efficacy of perineural dexamethasone as an adjunct for peripheral nerve block46. They found, likewise, a prolongation of postoperative analgesia. Dexamethasone increased the mean duration of analgesia by 233 minutes when combined with a short- or medium-term action local anaesthetic and by 488 (419–557) minutes when injected together with long-term action local anaesthetics (p <0.00001 for both). They could not see any clear dose response, the prolongation was not significantly dependent on the dose (4 to 10 mg dexamethasone), and it seems that intravenous and perineural administration has an equivalent effect. A fourth meta-analysis by Huynh et al. was recently published, in November 201547. In all, 1054 patients, 512 receiving perineural dexamethasone, from 12 trials were included. Ten were assessing the effects for brachial plexus nerve block. Doses of between 4 and 10 mg dexamethasone-containing local anaesthetic solutions showed a faster onset of action and resulted in a significant prolongation in the duration of analgesia (mean difference 351 minutes [P < 0.001] and motor blockade mean 277 minutes [P < 0.001]) compared with sole local anaesthetic. Time to onset of sensory and motor blocks was also significantly reduced with dexamethasone by about 1 minute. Dexamethasone significantly decreased postoperative nausea and vomiting by 9 vs. 27%. Thus, dexamethasone approximately doubled the duration of postoperative analgesia when it was combined with intermediate-acting (lidocaine and mepivacaine) or long-acting (bupivacaine and ropivacaine) local anaesthetics and had a clinically important effect in reducing postoperative nausea and vomiting.\n\nThere are sparse data around the addition/co-administration of buprenorphine for upper extremity blocks. In 2012, Behr et al. published a study assessing the perineural vs. systemic effects of buprenorphine in patients scheduled for shoulder arthroscopic surgery for a rotator cuff tear under middle interscalene brachial plexus block with 29.5 mL of 0.75% levobupivacaine48. The patients were randomised to receive additionally either saline or intramuscular buprenorphine 0.15 mg or epineural buprenorphine 0.15 mg. They found that the duration of both sensory block and postoperative analgesia was longer (P <0.05) in patients who had received epineural buprenorphine (856.1 ± 215.2 and 1049.7 ± 242.2 minutes) than in patients who had received intramuscular buprenorphine (693.6 ± 143.4 and 820.3 ± 335.3 minutes) or saline (488.3 ± 137.6 and 637.5 ± 72.1 minutes). The need for postoperative rescue analgesics was also lower in the epineural buprenorphine group than in the other two groups. Buprenorphine has also gained huge interest, and a recent paper by Kosel et al. suggested it is a “unique opioid adjuvant in regional anesthesia”49. Thus, available clinical data suggest that local anaesthesia combined with clonidine, buprenorphine, or dexamethasone has beneficial effects prolonging the analgesic effect and reducing pain and need for rescue medication and do not appear to alter local anaesthetic neurotoxicity50. There are, however, in vitro studies raising concern around neurotoxicity both by ropivacaine plain and when combined with midazolam and dexamethasone34. It should, however, be recalled that perineural administration of these compounds is, in most countries, still considered to be “off-label use”.\n\n\nPerineural catheters\n\nAn alternative option is to use an indwelling perineural catheter for continuous administration of local anaesthetic with or without adjuncts. Ullah et al. published a paper aimed at the performance of a meta-analysis of available studies assessing the continuous technique15. However, they were unable to make any analysis because of too few papers and heterogeneous design. They still commented that the continuous interscalene block seems to provide better pain relief as compared to parenteral opioids and to have a favourable safety profile when ultrasound technique was used. The improved safety using ultrasound-guided block technique and subsequent lower volume of local anaesthesia has been documented repeatedly51,52. It also facilitates success rate and shortens time to onset53. Ilfeld et al. have published two papers supporting the effective pain management with continuous perineural catheter in ambulatory shoulder surgery, showing significant effect on pain and discharge time54,55.\n\n\nFuture perspectives\n\nUpper extremity blocks provide effective analgesia for the duration of the local anaesthetic used. They provide an opioid-sparing effect during the early postoperative course up to 24 hours after surgery; however, the more protracted/long-term benefits are not extensively studied. The addition of adjuncts facilitates the duration and possibly reduces the risk of pain rebound when the analgesic effects wear off. It seems of importance to conduct further high-quality studies including more long-term recovery and outcomes56. Studies that compare the different blocks based on combination of local anaesthesia adjuncts that facilitate and extend the analgesia duration are required. There is also a need for further studies on mechanism of action, assessing whether the additive effect of adjuncts is local or merely systemic. The studies should be three armed with sole local anaesthesia, perineural adjunct/placebo systemic, and placebo perineural and adjunct systemic. Assessing not only the early first 24-hour postoperative pain effects but also the overall recovery and the effects for up to at least a week after surgery using validated recovery tools such as the Postoperative Quality of Recovery scale57 are warranted. Fischer and Bosch did indeed address this in a recent paper (Does regional anaesthesia improve outcome after surgery?), questioning the explicit benefits of the peripheral blocks unless combined with a dedicated enhanced recovery pathway program58.\n\n\nSummary and conclusion\n\nThe ultrasound technique has had a major impact on anaesthesiologists’ interest in performing upper extremity blocks. The increased interest in, training in, and use of the ultrasound technique have also reasonably improved the safety of the performance of the upper extremity, interscalene plexus, supraclavicular/infraclavicular as well as axillary plexus blocks. An update found evidence that upper extremity blocks provide effective intraoperative and early postoperative analgesia, reducing opioid consumption in the first 24 hours, but may cause rebound pain59,60. The addition of dexamethasone and/or alpha-2-agonist may improve the quality of and extend the duration of analgesia. However, further studies are warranted; assessing whether adjuncts have a local action or can be administered systemically and further assessing the effects of the block technique not only on early pain but also on the quality of recovery during the early and more protracted/long-term effects, beyond the first 24 to 48 hours, is warranted. The continuous perineural infusion is a feasible alternative, providing effective analgesia and improving quality of recovery, sleep quality, and patient satisfaction.\n\n\nKey message\n\nUpper extremity surgery can be performed safely in peripheral block as sole anaesthesia or in combination with sedation and general anaesthesia as needed.\n\nPlanning and proper logistics are of importance when implementing regional anaesthesia.\n\nTraining and skill in the regional anaesthesia technique must be secured.\n\nFactors of importance include time to onset of block, duration of surgical anaesthesia, and duration of postoperative analgesia.\n\nThe use of nerve stimulation or even more ultrasound guidance improves the success rate of the block and reduces the risk of side effects.\n\nUse of long-acting local anaesthetic solution delays onset but prolongs duration of effective anaesthesia.\n\nAddition of adjuncts prolongs the duration of postoperative analgesia, but further studies are warranted in order to better document safety and whether systemic administration is equally effective as compared to perineural administration.\n\nPerineural catheters and continuous infusion are also an effective alternative to prolong the analgesic effects.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nvan Zundert A, Helmstädter A, Goerig M, et al.: Centennial of intravenous regional anesthesia. Bier's Block (1908–2008). Reg Anesth Pain Med. 2008; 33(5): 483–9. PubMed Abstract | Publisher Full Text\n\nLong TR, Wass CT, Burkle CM: Perioperative interscalene blockade: an overview of its history and current clinical use. J Clin Anesth. 2002; 14(7): 546–56. PubMed Abstract | Publisher Full Text\n\nLabat G: Brachial Plexus Block: Some Details of Technique. Anesthesia & Analgesia. 1927; 6(2): 81–82. Publisher Full Text\n\nBenhamou D: Axillary plexus block using multiple nerve stimulation: a European view. Reg Anesth Pain Med. 2001; 26(6): 495–8. 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PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nThackeray EM, Swenson JD, Gertsch MC, et al.: Diaphragm function after interscalene brachial plexus block: a double-blind, randomized comparison of 0.25% and 0.125% bupivacaine. J Shoulder Elbow Surg. 2013; 22(3): 381–6. PubMed Abstract | Publisher Full Text\n\nStundner O, Meissnitzer M, Brummett CM, et al.: Comparison of tissue distribution, phrenic nerve involvement, and epidural spread in standard- vs low-volume ultrasound-guided interscalene plexus block using contrast magnetic resonance imaging: a randomized, controlled trial†. Br J Anaesth. 2016; 116(3): 405–12. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTran DQ, Russo G, Muñoz L, et al.: A prospective, randomized comparison between ultrasound-guided supraclavicular, infraclavicular, and axillary brachial plexus blocks. Reg Anesth Pain Med. 2009; 34(4): 366–71. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSunderland S, Yarnold CH, Head SJ, et al.: Regional Versus General Anesthesia and the Incidence of Unplanned Health Care Resource Utilization for Postoperative Pain After Wrist Fracture Surgery: Results From a Retrospective Quality Improvement Project. Reg Anesth Pain Med. 2016; 41(1): 22–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWilliams BA, Hough KA, Tsui BY, et al.: Neurotoxicity of adjuvants used in perineural anesthesia and analgesia in comparison with ropivacaine. Reg Anesth Pain Med. 2011; 36(3): 225–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKirksey MA, Haskins SC, Cheng J, et al.: Local Anesthetic Peripheral Nerve Block Adjuvants for Prolongation of Analgesia: A Systematic Qualitative Review. PLoS One. 2015; 10(9): e0137312. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBailard NS, Ortiz J, Flores RA: Additives to local anesthetics for peripheral nerve blocks: Evidence, limitations, and recommendations. Am J Health Syst Pharm. 2014; 71(5): 373–85. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSingelyn FJ, Gouverneur JM, Robert A: A minimum dose of clonidine added to mepivacaine prolongs the duration of anesthesia and analgesia after axillary brachial plexus block. Anesth Analg. 1996; 83(5): 1046–50. PubMed Abstract | Publisher Full Text\n\nMcCartney CJ, Duggan E, Apatu E: Should we add clonidine to local anesthetic for peripheral nerve blockade? A qualitative systematic review of the literature. Reg Anesth Pain Med. 2007; 32(4): 330–8. PubMed Abstract | Publisher Full Text\n\nPöpping DM, Elia N, Marret E, et al.: Clonidine as an adjuvant to local anesthetics for peripheral nerve and plexus blocks: a meta-analysis of randomized trials. Anesthesiology. 2009; 111(2): 406–15. PubMed Abstract | Publisher Full Text\n\nSwami SS, Keniya VM, Ladi SD, et al.: Comparison of dexmedetomidine and clonidine (α2 agonist drugs) as an adjuvant to local anaesthesia in supraclavicular brachial plexus block: A randomised double-blind prospective study. Indian J Anaesth. 2012; 56(3): 243–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbdallah FW, Brull R: Facilitatory effects of perineural dexmedetomidine on neuraxial and peripheral nerve block: a systematic review and meta-analysis. Br J Anaesth. 2013; 110(6): 915–25. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWu HH, Wang HT, Jin JJ, et al.: Does dexmedetomidine as a neuraxial adjuvant facilitate better anesthesia and analgesia? A systematic review and meta-analysis. PLoS One. 2014; 9(3): e93114. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKaur H, Singh G, Rani S, et al.: Effect of dexmedetomidine as an adjuvant to levobupivacaine in supraclavicular brachial plexus block: A randomized double-blind prospective study. J Anaesthesiol Clin Pharmacol. 2015; 31(3): 333–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChoi S, Rodseth R, McCartney CJ: Effects of dexamethasone as a local anaesthetic adjuvant for brachial plexus block: a systematic review and meta-analysis of randomized trials. Br J Anaesth. 2014; 112(3): 427–39. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDe Oliveira GS Jr, Castro Alves LJ, Nader A, et al.: Perineural dexamethasone to improve postoperative analgesia with peripheral nerve blocks: a meta-analysis of randomized controlled trials. Pain Res Treat. 2014; 2014: 179029. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlbrecht E, Kern C, Kirkham KR: A systematic review and meta-analysis of perineural dexamethasone for peripheral nerve blocks. Anaesthesia. 2015; 70(1): 71–83. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHuynh TM, Marret E, Bonnet F: Combination of dexamethasone and local anaesthetic solution in peripheral nerve blocks: A meta-analysis of randomised controlled trials. Eur J Anaesthesiol. 2015; 32(11): 751–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBehr A, Freo U, Ori C, et al.: Buprenorphine added to levobupivacaine enhances postoperative analgesia of middle interscalene brachial plexus block. J Anesth. 2012; 26(5): 746–51. PubMed Abstract | Publisher Full Text\n\nKosel J, Bobik P, Tomczyk M: Buprenorphine - the unique opioid adjuvant in regional anesthesia. Expert Rev Clin Pharmacol. 2016; 9(3): 375–83. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKnight JB, Schott NJ, Kentor ML, et al.: Neurotoxicity of common peripheral nerve block adjuvants. Curr Opin Anaesthesiol. 2015; 28(5): 598–604. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRiazi S, Carmichael N, Awad I, et al.: Effect of local anaesthetic volume (20 vs 5 ml) on the efficacy and respiratory consequences of ultrasound-guided interscalene brachial plexus block. Br J Anaesth. 2008; 101(4): 549–56. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRenes SH, Rettig HC, Gielen MJ, et al.: Ultrasound-guided low-dose interscalene brachial plexus block reduces the incidence of hemidiaphragmatic paresis. Reg Anesth Pain Med. 2009; 34(5): 498–502. PubMed Abstract | Publisher Full Text\n\nRanganath A, Srinivasan KK, Iohom G: Ultrasound guided axillary brachial plexus block. Med Ultrason. 2014; 16(3): 246–51. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nIlfeld BM, Vandenborne K, Duncan PW, et al.: Ambulatory continuous interscalene nerve blocks decrease the time to discharge readiness after total shoulder arthroplasty: a randomized, triple-masked, placebo-controlled study. Anesthesiology. 2006; 105(5): 999–1007. PubMed Abstract | F1000 Recommendation\n\nIlfeld BM, Morey TE, Wright TW, et al.: Continuous interscalene brachial plexus block for postoperative pain control at home: a randomized, double-blinded, placebo-controlled study. Anesth Analg. 2003; 96(4): 1089–95, table of contents. PubMed Abstract | Publisher Full Text\n\nJakobsson J: Assessing recovery after ambulatory anaesthesia, measures of resumption of activities of daily living. Curr Opin Anaesthesiol. 2011; 24(6): 601–4. PubMed Abstract | Publisher Full Text\n\nBowyer A, Jakobsson J, Ljungqvist O, et al.: A review of the scope and measurement of postoperative quality of recovery. Anaesthesia. 2014; 69(11): 1266–78. PubMed Abstract | Publisher Full Text\n\nFischer B, Bosch OD: Does regional anaesthesia improve outcome after surgery? Anaesthesia Intens Care Med. 2015; 16(11): 574–7. Publisher Full Text\n\nCandido KD, Knezevic NN: All adjuvants to local anesthetics were not created equal: animal data evaluating neurotoxicity, thermal hyperalgesia, and relevance to human application. Reg Anesth Pain Med. 2011; 36(3): 211–2. PubMed Abstract | Publisher Full Text\n\nSunderland S, Yarnold CH, Head SJ, et al.: Regional Versus General Anesthesia and the Incidence of Unplanned Health Care Resource Utilization for Postoperative Pain After Wrist Fracture Surgery: Results From a Retrospective Quality Improvement Project. Reg Anesth Pain Med. 2016; 41(1): 22–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13886",
"date": "18 May 2016",
"name": "Johan Raeder",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13887",
"date": "18 May 2016",
"name": "Paulo Lemos",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13888",
"date": "18 May 2016",
"name": "Stephen Choi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-907
|
https://f1000research.com/articles/4-690/v1
|
07 Sep 15
|
{
"type": "Research Note",
"title": "The possible importance of income and education as covariates in cohort studies",
"authors": [
"Norman Temple"
],
"abstract": "Background: Many cohort studies have been carried out that have provided information on the relationship between diet and health-related outcomes. Omission of important covariates during multivariate analysis may give rise to error due to residual confounding. A possibly important covariate is socioeconomic status (SES) as this is related to both diet and health.\nMethodology: An analysis was carried out of 76 randomly selected papers from 66 cohort studies. The papers covered many dietary variables and a wide variety of diseases/health-related outcomes. The cohort studies were carried out in many different locations and the subjects varied widely in age.\nResults: Approximately two-thirds of the papers (65.8%) used at least one measure of SES as a covariate. Education was used most often (60.5% of papers), followed by income (14.4%) and social class (2.6%). More than one measure of SES was used in 11.8% of papers.\nConclusions: Failure to include income (or another measure of present SES, such as occupation) may therefore be a common source of error in cohort studies. Failure to include education may be particularly important as it is likely to be a weaker measure of present SES than is income. There is a need for more research on this question. SES in childhood is almost never included in multivariate analysis in cohort studies carried out on adults. This could also play a significant role in disease risk in middle age or later. Very little is known regarding whether this is also a source of residual confounding.",
"keywords": [
"Cohort studies",
"socioeconomic status",
"covariates"
],
"content": "Introduction\n\nSocioeconomic status (SES) is related to diet and health. It is well established that affluent people have superior health compared to poorer people1. A major part of the explanation for this is that people with high SES generally follow a healthier lifestyle1. Of particular note, many studies have reported that people with a higher SES consume a more nutritious diet2. This pattern has been consistently reported in the USA, Canada, and many European countries, and is seen with different indicators of SES – education, income, and occupation.\n\nThis suggests that income, or other indicators of SES, may be a relevant factor in cohort studies. It follows, therefore, that failure to include SES in multivariate analysis of findings from cohort studies may be a source of error due to residual confounding. The potential importance of this was shown by an analysis of data from the British Women’s Heart and Health Study. Adjusting for a range of factors that indicate SES, both those in childhood and adulthood, attenuated the relationship between the plasma concentration of vitamins C and E and risk of coronary heart disease (CHD) in adults aged over 60 years3,4.\n\nThe analysis described here was carried out in order to investigate the extent of this potential source of error.\n\n\nMethods\n\nAn analysis was carried out of papers that reported the findings of cohort studies. The main inclusion criteria were, first, the papers were published in journals in the year 2000 or later, and, second, they reported findings on the relationship between dietary intake and health-related outcomes, such as body weight or disease. The papers were found using two main search strategies: (1) they were cited in various meta-analyses; and (2) by searching journals that cover the areas of nutrition, health, and medicine but with the search restricted to issues published in late 2013. A list of all papers used in this study is included in the Supplementary material.\n\nEach paper was studied and key information was extracted. In particular, a record was made indicating whether the risk ratios were adjusted for factors that indicate SES, such as education and income. Where available, information was also extracted that reported associations between SES and dietary variables (e.g., whether study subjects with more education consumed more fish). In addition, for comparative purposes a record was kept of whether alcohol, physical activity, and hypertension were included as covariates.\n\nMany cases were found where two or more papers were based on the same cohort study. In those cases each paper was evaluated. However, only one of the papers was included here unless, with respect to the covariates that are the focus of this paper, the papers used different covariates in the multivariate analysis. Papers were also included if they contained relevant information on the association between SES and dietary variables. After these exclusions and inclusions 76 papers from 66 cohort studies were included in the final analysis. Ten were published in the years 2000 to 2003, 15 during 2004 to 2007, 27 during 2008 to 2012, and 24 during 2013. The study is therefore based on a convenience sample of cohort studies rather than a systematic review. Nevertheless, because of the large number of cohort studies included, the findings are likely to be representative of cohort studies published in recent years.\n\n\nResults\n\nThe findings reported here are based on 66 cohort studies of which 52 were carried out on adults and 14 on children (age <18 years). They were carried out in the USA (30), Europe (28), Asia (5), and Canada (3).\n\nThe analysis included 76 papers. They covered a wide variety of diseases/health-related outcomes, including body weight or another measure of adiposity (25), cancer (21), type 2 diabetes (5), and all-cause mortality (16). Cardiovascular disease was studied in 33 papers, of which 19 looked at all forms of CVD combined, 13 at CHD, one at both CVD and CHD, and two at stroke. These 76 papers covered many aspects of the diet, including sugar-sweetened beverages (18), diet patterns (14), multivitamin supplements (8), fish (7), milk (7), sodium (6), and meat (4). The great majority of the papers covered only one dietary variable; the main exception was studies on beverages where different beverages were often included in the same paper. Physical activity was included as a covariate in 56 of the 76 papers (73.7%), alcohol in 43 of the 61 papers on adults (70.5%), and hypertension or blood pressure in 21 of the 33 papers on CVD (63.6%). At least one measure of SES was included as a covariate in 50 of the 76 papers (65.8%). Education was by far the most common (60.5%), followed by income (14.4%) and social class (2.6%). Several papers (11.8%) included more than one measure of SES.\n\nPapers based on studies of children used a measure of SES with a similar frequency to those on adults. Parental SES was used in those studies.\n\nMany of the papers reviewed here reported on the association between SES and diet. In general, people with more education consumed a diet that is associated with better health. This includes more fish5–7, less meat8,9, as well as a healthier overall dietary pattern10–13. In addition, some papers reported that those with more education consume more sugar-sweetened beverages14,15, more fruit juice14,16, and more sodium17,18. A particularly clear trend is the association between education and consumption of multivitamin supplements19–24.\n\nA small number of the papers reported on the association between income and diet. Consistent with the above findings higher income is associated with eating more fish6,7 and a healthier overall dietary pattern12.\n\n\nDiscussion\n\nThe methodology used here has strengths and weaknesses. The papers analyzed were mostly published in the last few years (51 of the 77 papers appeared in 2008 or later while the remainder appeared between 2000 and 2008). They cover a wide range of dietary components and outcomes. Sixty-four of the 66 cohort studies were carried out in the USA, Canada, Europe, or Japan. The findings are therefore likely to be representative of cohort studies published in recent years that investigated the relationship between diet and risk of a wide variety of diseases and health-related outcomes. However, as the papers analyzed were randomly selected, there may be a degree of selection bias.\n\nApproximately two-thirds of the papers (65.8%) used at least one measure of SES as a covariate. The most commonly used one was education (used in 60.5% of papers), followed by income (14.4%) and social class (2.6%). Some papers (11.8%) used more than one measure of SES. The frequency of inclusion of SES as a covariate is similar to that seen for the other covariates that were looked at here (73.7% for physical activity, 70.5% for alcohol in papers on adults, and 63.6% for hypertension or blood pressure in papers on CVD).\n\nMany of the papers provided information on the relationship between SES and diet. The findings reveal that people with more education typically consume a healthier diet. However, with some aspects of the diet the opposite trend was seen: several papers reported that those with more education consume more sugar-sweetened beverages and more sodium. They also consume more multivitamin supplements. Income also seems to be positively associated with diet quality; however, only a handful of papers provided information on this. That persons with a higher SES consume a generally more nutritious diet is consistent with previous studies (2). Likewise, previous studies have also consistently reported that users of multivitamin supplements are generally better educated25–27.\n\nThe findings reported here indicate that failure to include SES in multivariate analysis of cohort studies may be a source of error due to residual confounding. There are several ways by which this might happen. For example, in studies of CVD, error may occur if people of higher SES have better access to the health-care system and are therefore more likely to be screened for risk factors for CVD and to then receive better quality preventive treatment (such as diagnosis and treatment of hypertension). Similarly, error may arise in studies where body weight is the end-point if those of higher SES make greater efforts than those of lower SES to avoid excess weight gain. Conversely, lower SES may increase the risk of disease via psychological pathways, such as by raising the level of stress or by inducing feelings of disempowerment.\n\nIn some cohort studies subjects are fairly similar with respect to SES. For example, the Physicians' Health Study included only male physicians28. In such cases failure to adjust for SES is unlikely to lead to a significant error. However, in the large majority of cohort studies the subjects have a fairly wide variation in level of education and income.\n\nEducation, income, and social class (or occupation) has each been used as a covariate in the analysis of results from cohort studies. Education has been used far more often than the other two indicators of SES. While all three are closely associated with SES, they are quite distinct. For example, many people have a relatively poor education but still achieve a high income. This often occurs by marriage or by becoming a successful businessperson. Conversely, a person may have a college education but end up with a low income. It follows, therefore, that income (which indicates SES at the present time) may exert more influence on health and behavior than does education (which may have ceased 20 years or more before the cohort study started). Support for this possibility came from an Australian analysis that concluded that income was more strongly associated with diet than was education29. These findings indicate, therefore, that as only a small minority of cohort studies (14.4% of the publications) included income as a covariate, this may be a significant source of error due to residual confounding.\n\nA related question is SES in childhood as this could play a significant role in disease risk in middle age or later. This was suggested by findings from a British cohort study3,4. Adjusting for a range of factors that indicate SES, both those in childhood and adulthood, attenuated the relationship between the plasma concentration of both vitamins C and E and risk of CHD in adults aged over 60. With the exception of that study, I am unaware of any other cohort study that has examined the relationship between SES in childhood and disease risk in adults older than age 50.\n\nAs cohort studies vary widely in such features as the country where they are conducted, the age and gender of subjects, the dietary variables being studied, and the health outcomes being investigated, it is likely that the magnitude of the error caused by residual confounding will also be highly variable. More research effort is required to determine the extent to which failure to adjust for SES across the lifecycle (as well as of other covariates) is a source of error in cohort studies.\n\nAnother potentially important covariate is growth in the fetal period and early infancy. Fetal development, as indicated by birthweight, as well as the rate of weight gain in early childhood, are predictors of risk of developing heart disease and type 2 diabetes 40 or 50 years later30. While these variables may be only weakly related to SES, they are relevant here as they again underscore how diet, lifestyle, and SES in childhood (or even before birth) may be associated with disease risk decades later.\n\nIn conclusion, many cohort studies may have residual confounding caused by the failure to adjust for key covariates. Little attention seems to have been paid to this possible source of significant error. Of particular concern is the question of SES: roughly one third of papers have not included any measure of SES among the covariates. Education was used as the measure of SES far more often than income. However, education is likely to be a weaker measure of present SES than is income. There is a need for more research on the extent to which failure to include income (or another measure of present SES, such as occupation) leads to error in cohort studies.",
"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\nSupplementary material\n\nPapers used as a source of information for this study.\n\nPapers with the same number but different letters (e.g., 2a and 2b) indicate that the same cohort study contributed two separate papers\n\nClick here to access the data.\n\n\nReferences\n\nDi Cesare M, Khang YH, Asaria P, et al.: Inequalities in non-communicable diseases and effective responses. Lancet. 2013; 381(9866): 585–597. PubMed Abstract | Publisher Full Text\n\nDarmon N, Drewnowski A: Does social class predict diet quality? Am J Clin Nutr. 2008; 87(5): 1107–1117. PubMed Abstract\n\nLawlor DA, Davey Smith G, Kundu D, et al.: Those confounded vitamins: what can we learn from the differences between observational versus randomised trial evidence? Lancet. 2004; 363(9422): 1724–1727. PubMed Abstract | Publisher Full Text\n\nLawlor DA, Ebrahim S, Kundu D, et al.: Vitamin C is not associated with coronary heart disease risk once life course socioeconomic position is taken into account: prospective findings from the British Women’s Heart and Health Study. Heart. 2005; 91(8): 1086–1087. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Goede J, Geleijnse JM, Boer JM, et al.: Marine (n-3) fatty acids, fish consumption, and the 10-year risk of fatal and nonfatal coronary heart disease in a large population of Dutch adults with low fish intake. J Nutr. 2010; 140(5): 1023–1028. PubMed Abstract | Publisher Full Text\n\nTomasallo C, Anderson H, Haughwout M, et al.: Mortality among frequent consumers of Great Lakes sport fish. Environ Res. 2010; 110(1): 62–69. PubMed Abstract | Publisher Full Text\n\nFolsom AR, Demissie Z: Fish intake, marine omega-3 fatty acids, and mortality in a cohort of postmenopausal women. Am J Epidemiol. 2004; 160(10): 1005–1010. PubMed Abstract | Publisher Full Text\n\nSinha R, Cross AJ, Graubard BI, et al.: Meat intake and mortality: a prospective study of over half a million people. Arch Intern Med. 2009; 169(6): 562–571. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRohrmann S, Overvad K, Bueno-de-Mesquita HB, et al.: Meat consumption and mortality--results from the European Prospective Investigation into Cancer and Nutrition. BMC Med. 2013; 11: 63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoggs DA, Rosenberg L, Rodríguez-Bernal CL, et al.: Long-term diet quality is associated with lower obesity risk in young African American women with normal BMI at baseline. J Nutr. 2013; 143(10): 1636–1641. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWengreen H, Munger RG, Cutler A, et al.: Prospective study of Dietary Approaches to Stop Hypertension- and Mediterranean-style dietary patterns and age-related cognitive change: the Cache County Study on Memory, Health and Aging. Am J Clin Nutr. 2013; 98(5): 1263–1271. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParrott MD, Shatenstein B, Ferland G, et al.: Relationship between diet quality and cognition depends on socioeconomic position in healthy older adults. J Nutr. 2013; 143(11): 1767–1773. PubMed Abstract | Publisher Full Text\n\nVelie EM, Schairer C, Flood A, et al.: Empirically derived dietary patterns and risk of postmenopausal breast cancer in a large prospective cohort study. Am J Clin Nutr. 2005; 82(6): 1308–1319. PubMed Abstract\n\nVanselow MS, Pereira MA, Neumark-Sztainer D, et al.: Adolescent beverage habits and changes in weight over time: findings from Project EAT. Am J Clin Nutr. 2009; 90(6): 1489–1495. PubMed Abstract | Publisher Full Text\n\nPalmer JR, Boggs DA, Krishnan S, et al.: Sugar-sweetened beverages and incidence of type 2 diabetes mellitus in African American women. Arch Intern Med. 2008; 168(14): 1487–1492. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnson L, Mander AP, Jones LR, et al.: Is sugar-sweetened beverage consumption associated with increased fatness in children? Nutrition. 2007; 23(7–8): 557–563. PubMed Abstract | Publisher Full Text\n\nCohen HW, Hailpern SM, Fang J, et al.: Sodium intake and mortality in the NHANES II follow-up study. Am J Med. 2006; 119(3): 275.e7–14. PubMed Abstract | Publisher Full Text\n\nCohen HW, Hailpern SM, Alderman MH: Sodium intake and mortality follow-up in the Third National Health and Nutrition Examination Survey (NHANES III). J Gen Intern Med. 2008; 23(9): 1297–1302. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi K, Kaaks R, Linseisen J, et al.: Vitamin/mineral supplementation and cancer, cardiovascular, and all-cause mortality in a German prospective cohort (EPIC-Heidelberg). Eur J Nutr. 2012; 51(4): 407–413. PubMed Abstract | Publisher Full Text\n\nMursu J, Robien K, Harnack LJ, et al.: Dietary supplements and mortality rate in older women: the Iowa Women’s Health Study. Arch Intern Med. 2011; 171(18): 1625–1633. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeuhouser ML, Wassertheil-Smoller S, Thomson C, et al.: Multivitamin use and risk of cancer and cardiovascular disease in the Women’s Health Initiative cohorts. Arch Intern Med. 2009; 169(3): 294–304. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPark SY, Murphy SP, Wilkens LR, et al.: Multivitamin use and the risk of mortality and cancer incidence: the multiethnic cohort study. Am J Epidemiol. 2011; 173(8): 906–914. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRautiainen S, Akesson A, Levitan EB, et al.: Multivitamin use and the risk of myocardial infarction: a population-based cohort of Swedish women. Am J Clin Nutr. 2010; 92(5): 1251–1256. PubMed Abstract | Publisher Full Text\n\nWatkins ML, Erickson JD, Thun MJ, et al.: Multivitamin use and mortality in a large prospective study. Am J Epidemiol. 2000; 152(2): 149–162. PubMed Abstract | Publisher Full Text\n\nRadimer K, Bindewald B, Hughes J, et al.: Dietary supplement use by US adults: data from the National Health and Nutrition Examination Survey, 1999–2000. Am J Epidemiol. 2004; 160(4): 339–349. PubMed Abstract | Publisher Full Text\n\nMillen AE, Dodd KW, Subar AF: Use of vitamin, mineral, nonvitamin, and nonmineral supplements in the United States: The 1987, 1992, and 2000 National Health Interview Survey results. J Am Diet Assoc. 2004; 104(6): 942–950. PubMed Abstract | Publisher Full Text\n\nSatia-Abouta J, Kristal AR, Patterson RE, et al.: Dietary supplement use and medical conditions: the VITAL study. Am J Prev Med. 2003; 24(1): 43–51. PubMed Abstract | Publisher Full Text\n\nHshieh TT, Petrone AB, Gaziano JM, et al.: Nut consumption and risk of mortality in the Physicians’ Health Study. Am J Clin Nutr. 2015; 101(2): 407–412. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTurrell G, Hewitt B, Patterson C, et al.: Measuring socio-economic position in dietary research: is choice of socio-economic indicator important? Public Health Nutr. 2003; 6(2): 191–200. PubMed Abstract | Publisher Full Text\n\nBarker DJP: The developmental origins of chronic disease in later life. In: Temple NJ, Wilson T, Jacobs DR, eds. Nutritional Health: Strategies for Disease Prevention, 3rd ed. New York: Humana Press, 2012; 59–83. Publisher Full Text"
}
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[
{
"id": "11155",
"date": "18 Nov 2015",
"name": "Tord Finne Vedøy",
"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 an important reminder that SES is a crucial factor to consider when doing research on health related lifestyle issues. However, I do have some comments of minor importance.First, in the first paragraph of the introduction the author emphasizes the role of a nutritious diet for a health lifestyle. Although I understand that the paper focuses on nutrition, it could be noted that smoking still is one of the largest, if not the largest, single cause of death and disease in most developed countries. For the same reasons it might be a good idea to mention in the title that the focus of the paper is nutrition.Second, the author should make explicit what is meant by “cohort studies”. Does this refer to panel-studies, age-period-cohort studies or both?Third, the transition from the first to the second paragraph in the introduction seems wanting. As I read it, it is the pattern (“that people with a higher SES consume a more nutritious diet”) that “suggests that income, or other indicators of SES, may be a relevant factor in cohort studies”. However, if cohort is to be an important factor, the temporal or historical dimension of the pattern should be emphasized.Fourth, the discussion of the relative importance of different measures of SES on health could be more nuanced. While there may be good reasons for using income rather than education as a measure of SES, especially in matters of consumption, education has certain characteristics which income lacks. For example, education is stable throughout the lifespan and provides individuals with certain abilities related to learning and thinking which are important in matters of health. These ideas has been discussed by for example Cutler and Lleras-Muney (2010).",
"responses": [
{
"c_id": "1965",
"date": "18 May 2016",
"name": "Norman Temple",
"role": "Author Response",
"response": "I thank Dr Vedøy for his helpful comments. I have made edits to the paper based on those comments."
}
]
},
{
"id": "13500",
"date": "12 May 2016",
"name": "Silvano Gallus",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written manuscript providing information on the use of socioeconomic status (SES) measures as covariates in 76 papers from 66 cohort studies. The study is interesting and original. I only have a few minor points which could improve the presentation of findings:The objectives of the study may be clarified in the background or methods sections of the abstract;In the conclusions of the abstract, I suggest to delete the term \"therefore\" (2nd line of the Conclusions);In population-based cohort studies, income may represent a major problem, given the likely high nonresponse rate. For example, in a European cross-sectional study I conducted in 2010 on more than 18,000 European adults, 22% of participants did not provide information on income, and only a negligible proportion of subjects did not provide information on education. Also for this reason, education (or alternatively occupation) may be considered a more appropriate proxy of SES than income. This issue may be further addressed both in the abstract and in the main text.",
"responses": [
{
"c_id": "3052",
"date": "25 Sep 2017",
"name": "Dr. Sindhu B.M.",
"role": "Reader Comment",
"response": "Respected Sir, I have a doubt regarding this. I request you to throw some light on this.. When people dont respond to a question (eg: income/education) in a study, should we take them as ‘non-responders’ and not analyse their data? Or analyse their data also?"
}
]
}
] | 1
|
https://f1000research.com/articles/4-690
|
https://f1000research.com/articles/5-893/v1
|
16 May 16
|
{
"type": "Review",
"title": "Network science for the identification of novel therapeutic targets in epilepsy",
"authors": [
"Rod C. Scott"
],
"abstract": "The quality of life of children with epilepsy is a function of seizures and associated cognitive and behavioral comorbidities. Current treatments are not successful at stopping seizures in approximately 30% of patients despite the introduction of multiple new antiepileptic drugs over the last decade. In addition, modification of seizures has only a modest impact on the comorbidities. Therefore, novel approaches to identify therapeutic targets that improve seizures and comorbidities are urgently required. The potential of network science as applied to genetic, local neural network, and global brain data is reviewed. Several examples of possible new therapeutic approaches defined using novel network tools are highlighted. Further study to translate the findings into clinical practice is now required.",
"keywords": [
"epilepsy",
"therapeutic targets",
"network science"
],
"content": "Introduction\n\nChildren with epilepsy have seizures that are frequently associated with cognitive and behavioral impairments1. In combination, these factors negatively impact quality of life2 and greatly diminish the ability of children with epilepsy to develop into successful adults. It is therefore critical that treatments aimed at modifying adverse outcomes are developed. Current therapies are largely targeted toward seizures and include antiepileptic drugs and dietary and surgical therapies. Although these therapies are effective in the treatment of seizures, approximately 30% of patients continue to have seizures despite maximal therapy3. Specific therapies for behavioral difficulties include stimulants4,5, antipsychotics6,7, and antidepressants6,7. Therapies for learning impairments are largely educational. In all of these cases, the therapy targets symptoms without modifying the brain disorder responsible for the seizures and the associated morbidities. In order to develop new therapies, it is important that mechanisms of the underlying brain disorders are understood so that therapies can be targeted towards modifying those abnormalities and thereby improve outcomes.\n\nThe mechanisms underpinning adverse outcomes can be studied at a variety of levels including the level of genes, receptors, cell signaling pathways, and networks. Network science is providing new ways of understanding disease mechanisms and could lead to novel therapies targeted directly at modifying network abnormalities underlying seizures and comorbidities, irrespective of the genetic or cell signaling abnormalities8 (Figure 1). This review will explore the potential of network science to contribute to the identification of novel therapeutic targets.\n\nEach node in the network could represent a gene, transcriptional module, single neuron, or brain region. The lines between the nodes represent relevant interactions between the nodes. Conceptually, the structure of the network is different in disease states compared to controls. In this example, the diseased network is over-connected (e.g. hypersynchrony in an epileptic brain). Targeting the network directly using drugs, brain stimulation, cell therapy, or transcranial magnetic stimulation approaches could modify the diseased network in order to allow more normal function. It remains unknown whether restoration of networks to normal (left network post-therapy) or modification to a network with similar phenotype to normal (right network post-therapy) is required. Understanding of these system-level mechanisms could lead to new treatments or optimization of currently used clinical tools, such as deep brain stimulation.\n\n\nUnderstanding disease mechanisms at network levels\n\nDisease mechanisms in the context of epilepsy can be conceptualized in at least two ways. The first is encapsulated in the epileptic encephalopathy hypothesis, which states that epileptic activity causes cognitive and behavioral impairments over and above those expected for the underlying etiology9. In this circumstance, it can be argued that epileptic activity itself modifies neural networks in a way that no longer supports normal cognitive and behavioral function. The therapeutic implication is that treatment of seizures and interictal discharges will positively modify adverse outcomes. Despite many years of trying to improve cognitive and behavioral outcomes by targeting seizures, the positive impacts are rather modest1,10,11. A common suggestion is that the treatment of seizures prevents any further decline in abilities. If correct, this suggests that alternative approaches to improving outcomes are required.\n\nThe second concept places etiology in the forefront. In this view, patients with epilepsy all have an underlying etiology (even if it is unrecognized) and this is directly responsible for the neural network disruptions that lead to the symptoms of seizures and adverse cognitive and behavioral outcomes. It remains possible that seizures also make a contribution to the modification of networks, although it is difficult to clearly identify the magnitude of the contribution12,13. However, if etiology is the major contributor, then treatment of seizures is unlikely to have major positive impact, as observed in clinical practice. If seizures and cognitive/behavioral impairments are primarily a function of etiology, then disease-modifying therapies would be expected to improve all of the symptoms. Although this is an area receiving increasing attention, no such therapies are yet available in clinical practice.\n\nThe application of network science to these issues is gaining popularity8. Network science is a mixture of graph theory, dynamical systems theory, and ideas from statistical mechanics8. These tools can be applied to genetic, electrophysiological, and magnetic resonance data. The appropriate application of network science to epilepsy requires an interdisciplinary approach involving clinicians, translational neuroscientists, computer scientists, and mathematicians. Examples of novel insights that are arising from these interdisciplinary collaborations are discussed below.\n\n\nGene regulation networks\n\nGenetic approaches have made significant contributions to the understanding of neurological disorders associated with seizures14–17. The hope is that gene replacement therapy may restore function, as in an animal model of Rett syndrome18. This approach relates to single gene disorders, and the list of genes associated with epilepsies is rapidly growing17,19. Although it may be possible to replace the identified genes, many abnormalities are extremely rare, raising an important concern about the practical use of such approaches. An alternative approach is to use systems genetics to characterize the genetic regulation of pathophysiological pathways. This approach has previously been used to explore the pathophysiology of type 1 diabetes20, autism21,22, febrile convulsions23–25, and the latter’s association with mesial temporal lobe epilepsy24. An example of how systems genetics could identify a therapeutic target in epilepsy comes from a study of temporal lobe epilepsy26. Johnson et al. evaluated gene expression networks in post-surgical hippocampal tissue from patients with temporal lobe epilepsy. They identified a specialized, highly expressed transcriptional module encoding proconvulsive cytokine and Toll-like receptor signaling genes. Sestrin 3 positively regulates this module in macrophages, microglia, and neurons. This finding is also present in pilocarpine-exposed mice, which demonstrated pilocarpine-induced status epilepticus with subsequent temporal lobe epilepsy, giving further credibility to the clinical finding. Importantly, knockdown of Sestrin 3 in zebrafish attenuates chemically induced behavioral seizures. Therefore, it is possible that modulation of Sestrin 3 could modulate seizures in humans. It is unlikely that this target would have been identified without the mathematical approaches inherent in systems genetics. This study used tissue from epilepsy surgery, was carried out in adults, and targeted an etiology common in adults. However, the pathophysiological processes leading to mesial temporal sclerosis (MTS) frequently occur during development. Children with epilepsies frequently have malformations of cortical development, and similar approaches could be applied to this surgical tissue and have potential to identify other novel therapeutic targets. Modification of genetic signaling can be achieved with small interfering RNA (siRNA) and microRNA (miRNA) approaches. Identification of specific genes or regulatory gene networks could provide critical targets for these approaches that are already being tested in the clinical domain27. These approaches can also be applied to epigenetic data and could provide important insights in that domain as well.\n\n\nLocal neural networks\n\nAnother level at which network approaches may identify therapeutic targets is at the level of local neuronal networks. This requires the identification of system-level mechanisms at the level of changes in neural circuitry that directly impair the ability of that circuit to support cognitive behavior. Information processing in a neural system is a function of the rate of firing of neurons (rate coding)28–32, the precise timing of action potential firing with respect to oscillatory activity (temporal coding)33,34, and joint activation of ensembles of neurons (population coding)35–38. It is known that each of these components of information processing can be abnormal in a variety of animal models of epilepsies. Rate coding as described by hippocampal place cells is disrupted following early life seizures and in the context of MTS39–42. The spatial fidelity of these cells is reduced and this is consistent with the abnormalities in spatial cognition identified in these models41,43–45. Temporal and population coding have also been evaluated in models of MTS and found to be abnormal36,39. For example, excessive synchrony of CA1 pyramidal cell firing predicts poor performance in a spatial alternation task, suggesting that the organization of neuronal firing within a neural population is important for behavior36. It remains uncertain whether this excessive synchrony is important for seizure generation, but modulation of this synchrony could both reduce seizures and improve cognition.\n\nMany of the epilepsy models described above have associated structural abnormalities. Remarkably, cognitive outcomes can be improved with environmental enrichment and overtraining strategies, even in the context of the structural brain abnormalities46, suggesting that structure-function relationships are not fixed. This could be exploited for therapeutic advantage if the network-level changes associated with these improvements could be identified. This remains an area of active research. However, some system-level interventions have been shown to improve outcomes in epilepsy. In humans, stimulation devices are known to reduce seizures and there is some evidence that stimulation of the entorhinal cortex can improve cognition47. A detailed understanding of how networks need to be modified to improve outcomes would allow optimization of these tools. Electrical stimulation has the potential downside of activating all cell types, which may limit the possibility of more subtly altering neuronal interactions. In the future, optogenetic stimulation may allow more precise modulation of neural network behavior by activating only certain cell types.\n\nInterneurons are critical for the organization of pyramidal cells48 and therefore may represent a cell target in epilepsy. Implantation of interneuron precursors into animal models of MTS reduces seizures49–51 and, at least in some experiments, improves cognitive outcomes49. In addition, optogenetic stimulation of interneurons using a closed loop system also reduces seizures52. The system-level mechanisms underlying the improvements have not yet been explored in these models, but such studies could ultimately provide essential information for optimizing parameters and maximizing outcomes.\n\nAlthough stimulation approaches are the most obvious application of the sort of system-level information described above, drug interventions may also be possible. For example, adrenocorticotropic hormone (ACTH) administration to rodents exposed to early life seizures improves attentional outcomes despite having no effect on the seizures53. This suggests that ACTH is modulating information processing in a way that is currently unknown but deserves future study. The implication for the treatment of patients is that medications that do not influence seizures but improve cognitive outcomes may be beneficial. Therefore, outcome measures for clinical trials may need to include cognitive outcomes, such as in the UKISS trial of infantile spasms54, and may even have to be designed to have cognition as the main outcome measure. Another speculative drug approach is the use of Designer Receptors Exclusively Activated by Designer Drugs (DREADDS)52. This approach may also allow targeting of certain cell types within a network in order to modify the population activity of that network.\n\n\nGlobal networks\n\nThe study of global networks within the context of epilepsy has used magnetic resonance, electroencephalography, and magnetoencephalography approaches. Although there is a wealth of data on the phenomenology of connectivity (see for example 55–59), there are far less data on how this information can be used for therapeutic purposes. However, an understanding of distributed networks could identify regions of the brain that are critical for seizure generation or for any relevant cognitive function, even if that area of the brain is not the obviously abnormal piece of tissue. An excellent example of this is from a study of patients with periventricular nodular heterotopia associated with seizures60. Functional MRI was used to identify aberrant functional connectivity between the heterotopia and normal-looking cortex. Abnormal connectivity was identified between the heterotopia and areas of the cortex that were not consistent across patients. Transcranial magnetic stimulation (TMS) was used to show that the connected cortical areas were hyperexcitable, suggesting that the authors had identified an epileptogenic network. This raises the possibility that TMS targeted at the cortical site identified using network approaches could reduce seizures in patients with deep-seated epileptogenic foci.\n\nAnother potential use of network approaches is in epilepsy surgery. Despite state-of-the-art selection methods, investigations for identifying the epileptogenic focus, and excellent surgical technique, many patients with epilepsy fail to become seizure free. In these patients, it is possible that the epileptogenic networks have not been adequately disrupted by surgery and therefore a more detailed understanding of the network could lead to tailored resections based on that information. A variety of network measures have been used to describe the distributed abnormal networks in patients with focal epilepsy, particularly those with MTS55,61–63. Patients who subsequently failed to become seizure free often had different network changes to those patients who did become seizure free. The next challenge is to establish how that information can be used to maximize surgical outcomes.\n\n\nConclusion\n\nThe goal of this article was not to provide an exhaustive review of network science as applied to epilepsy but rather to provide examples of how these methods have enormous potential to contribute to the treatment of patients with epilepsy. The overriding concept is that an understanding of networks from genetic, local neural, and global brain data can identify new therapeutic targets for both seizures and associated comorbidities. Modification of these targets can be with drugs, stimulation devices, surgical approaches, and TMS, ultimately maximizing the quality of life of patients with epilepsy.",
"appendix": "Competing interests\n\n\n\nThe author declares that he has no competing interests.\n\n\nGrant information\n\nThis work was supported by National Institutes of Health grant 1R01NS074689 to RS.\n\n\nReferences\n\nRaspall-Chaure M, Neville BG, Scott RC: The medical management of the epilepsies in children: conceptual and practical considerations. Lancet Neurol. 2008; 7(1): 57–69. PubMed Abstract | Publisher Full Text\n\nReilly C, Atkinson P, Das KB, et al.: Factors associated with quality of life in active childhood epilepsy: a population-based study. Eur J Paediatr Neurol. 2015; 19(3): 308–13. PubMed Abstract | Publisher Full Text\n\nKwan P, Brodie MJ: Early identification of refractory epilepsy. N Engl J Med. 2000; 342(5): 314–9. 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PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKrook-Magnuson E, Armstrong C, Oijala M, et al.: On-demand optogenetic control of spontaneous seizures in temporal lobe epilepsy. Nat Commun. 2013; 4: 1376. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHernan AE, Alexander A, Lenck-Santini PP, et al.: Attention deficit associated with early life interictal spikes in a rat model is improved with ACTH. PLoS One. 2014; 9(2): e89812. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDarke K, Edwards SW, Hancock E, et al.: Developmental and epilepsy outcomes at age 4 years in the UKISS trial comparing hormonal treatments to vigabatrin for infantile spasms: a multi-centre randomised trial. Arch Dis Child. 2010; 95(5): 382–6. 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PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nVega-Zelaya L, Pastor J, de Sola RG, et al.: Disrupted Ipsilateral Network Connectivity in Temporal Lobe Epilepsy. PLoS One. 2015; 10(10): e0140859. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "13863",
"date": "16 May 2016",
"name": "Jerome Engel Jr",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13864",
"date": "16 May 2016",
"name": "Guillermo J Ortega",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-893
|
https://f1000research.com/articles/5-892/v1
|
16 May 16
|
{
"type": "Review",
"title": "Intravital imaging reveals new ancillary mechanisms co-opted by cancer cells to drive tumor progression",
"authors": [
"Claire Vennin",
"David Herrmann",
"Morghan C. Lucas",
"Paul Timpson",
"Claire Vennin",
"David Herrmann",
"Morghan C. Lucas"
],
"abstract": "Intravital imaging is providing new insights into the dynamics of tumor progression in native tissues and has started to reveal the layers of complexity found in cancer. Recent advances in intravital imaging have allowed us to look deeper into cancer behavior and to dissect the interactions between tumor cells and the ancillary host niche that promote cancer development. In this review, we provide an insight into the latest advances in cancer biology achieved by intravital imaging, focusing on recently discovered mechanisms by which tumor cells manipulate normal tissue to facilitate disease progression.",
"keywords": [
"Intravital imaging",
"tumor progression"
],
"content": "Introduction\n\nWithin tumors, intricate crosstalk between cancer cells and the surrounding microenvironment supports cancer initiation and progression. Importantly, these interactions not only shape the development of the primary tumor but also are required at secondary sites to develop a microenvironment permissive to metastatic growth. This appreciation of the dynamic cancer-stroma interactions in tumor progression has led to a transition from traditional in vitro assays to more complex in vivo models to faithfully embrace the intricacy of cancer. In these models, intravital imaging has been used to dissect the molecular events governing cancer progression and revealed unprecedented information on the behavior of cells in their native environment, both within primary tumors and at distant metastatic sites. In combination with traditional and static approaches used in cancer research, intravital imaging has significantly expanded our understanding of the complexity of cancer biology, as it allows us to directly image the spatiotemporal dynamics of cancer progression in live settings and at the whole-organ, cellular, subcellular, and molecular levels. Here, we outline the latest discoveries facilitated by intravital imaging of tumors, which could not otherwise be achieved in vitro, and discuss how intravital imaging techniques used in other disease contexts could be repurposed for cancer research.\n\n\nImaging the tumor vasculature to study cancer growth and dissemination\n\nCancer cells use and co-opt the surrounding stroma to promote their expansion and dissemination. Tumor-associated blood vessels serve a dual function during cancer development: they provide tumor tissue with essential oxygen and nutrients but can also act as carriers for circulating cancer cells. While various models have been developed in vitro for studying microvasculature systems1,2, several intravital imaging tools, such as fluorescent proteins or nanoparticles, have been employed to understand the interactions between cancer cells and blood vessels in live settings, an aspect that cannot be recapitulated in vitro3–6. For example, fluorescently labeled lectins, such as Lens culinaris agglutinin, have been developed to selectively bind to endothelial cells3,4. Intravenous co-injection of agglutinin and fluorescently labeled cancer cells was recently used with intravital imaging of the embryonic chicken chorioallantoic membrane (CAM). This is a surrogate assay for the study of cancer cell extravasation and metastatic colonization and has recently provided insight into the time course and molecular mechanisms involved in cancer cell extravasation3,4. Here, the authors demonstrated that intravascular cancer cells initially move along the luminal endothelium in an amoeboid manner and subsequently start to extend protrusions in-between endothelial cells into the extravascular stroma. Cancer cells then gradually push through endothelial cell-cell junctions, in some cases even displacing endothelial cells4. Interestingly, invadopodia marker proteins such as cortactin or MT1-MMP were shown to be localized at the protrusions of extravasating cancer cells, and knockdown of these markers impaired cell extravasation and the formation of metastatic colonies within the CAM4. In line with this, tail-vein injection of invadopodia-deficient cells into mice resulted in a decreased number of lung metastases, showing that invadopodia are a key feature for transendothelial cancer cell migration4. Similarly, providing intravascular cancer cells in the CAM assay with hyaluronic acid, which is a component of the extracellular matrix (ECM) overexpressed in many tumor types7–9, led to increased cell extravasation3. Using this probe therefore demonstrated that the ECM not only represents a physical barrier against chemotherapy delivery10 but can also be co-opted by cancer cells for secondary site colonization.\n\nFluorescent dextrans and quantum dot (QD) nanoparticles are also commonly used to visualize blood vessels. More recently, they have been employed during in vivo cancer research to investigate vascular integrity in live tumor tissues11,12. For example, intravital time-course imaging of QDs in the MMTV-PyMT mouse model of breast cancer allowed Ormandy and colleagues to demonstrate that cancer cells at the primary site can use the surrounding blood vasculature during the progression of invasive and metastatic cancer13. Here, ELF5 induction resulted in QD leakage and accumulation within the interstitial space, in line with the formation of hemorrhagic tumors, and increased the number of lung metastases (Figure 1A, blood vessels in red, note the signal in areas around leaky vessels in PyMT/ELF5 mice13). Furthermore, we previously used QDs to map the vascularization of pancreatic subcutaneous xenograft tumors and showed in vivo that Src activity and drug-mediated Src inactivation in cancer cells correlate with their position relative to intratumoral blood vessels11. New organic QDs with improved biocompatibility, stability, and two-photon absorption cross-section and fluorescently tagged viral nanoparticles have recently been developed for real-time intravital mapping of the blood vasculature in vivo14,15. Fluorescently labeled red blood cells and mice with fluorescently labeled endothelial cells were also engineered to mark the blood vasculature in vivo16–18, and together with QDs and dextran, these approaches are likely to improve our understanding of how the tumor-associated vasculature supports cancer progression.\n\nA. Quantum dot (QD) imaging of angiogenesis and leaky tumor vasculature. Adapted from 13. B. Tracking of photo-converted neutrophils migrating through lymphatic networks. Adapted from 38. C. Identification of dormant myeloma cells homed into the bone niche. Adapted from 48. D. Direct visualization of local and systemic transfer of extracellular vesicles and exosomes between different cellular compartments. Adapted from 60. E. Second harmonic generation (SHG) analysis of collagen crosslinking to identify new regulators of extracellular matrix (ECM)-driven aggressiveness. Adapted from 74. F. Longitudinal monitoring of chemotherapy pharmacokinetics and targeting through an optical window. Adapted from 93. G. Dissection of spatiotemporal dynamics of molecular events driving cancer invasion and dissolution in native tissues using reporter mice. Adapted from 96 and 103. Abbreviations: CAF, cancer-associated fibroblast; GFP, green fluorescent protein.\n\nThe lymphatic system presents another route of cancer cell dissemination. For example, Das et al. recently used intravital imaging of melanoma cells injected into the mammary fat pad with a lymphatic tracer dye to visualize cancer cell migration towards tumor-draining lymph nodes19. The authors demonstrated that lymphatic sinuses of the lymph nodes, but not peripheral lymphatics, secrete CCL8, which activates CCR1 on the cancer cell compartment to permit entry into the lymph node19. Inhibition of CCR1 did not impair primary tumor cells from entering the lymphatic system but blocked their exit into the lymph node and arrested the circulating cells within nearby lymphatic vessels. This indicates that lymphatic cancer cell dissemination not only occurs through passive transport but also is supported by paracrine chemotactic signaling19. Recently, a transgenic reporter of lymphatic vessels was generated by crossing LSL-tdTomato mice with Prox1-Cre-ERT2 mice to induce fluorescent reporter expression in lymphatic vessels and map cell trafficking within lymphatic networks20. While the authors used this model to track dendritic cells entering tdTomato-expressing lymphatic vessels upon acute inflammation in vivo, this technology could be repurposed for future use in cancer research to watch circulating tumor cells disseminating through the lymphatic system.\n\n\nImaging the tumor-associated immune system\n\nDuring cancer progression, tumor cells interact with and manipulate the immune system to facilitate their growth and escape from cell death21–24. Here, dual intravital imaging of leaky blood vessels and immune cells has been used to identify novel mechanisms of drug action. In particular, the antitumor activity of bisphosphonates, which are commonly used in skeletal diseases, such as osteoporosis25, has been explored in a 4T1 mammary tumor mouse model26. Here, it was demonstrated that fluorescently labeled bisphosphonates enter the primary tumor site via the leaky vasculature and bind to microcalcifications within the tissue. Bisphosphonates were then incorporated by tumor-associated macrophages (TAMs), and the authors suggest that this might impair the function of TAMs26, which have been shown to potentiate tumor progression27. Thus, intravital imaging allowed the authors to delineate an unknown mechanism of action underlying the partial benefits of bisphosphonate treatment in breast cancer patients. In line with this, TAMs have also been identified in the tumor microenvironment of metastasis (TMEM), a hotspot of vascular permeability characterized by the physical interaction among tumor cells, TAMs, and endothelial cells28. Intravital imaging of blood vasculature using QDs and fluorescent dextran demonstrated that Tie2hi-expressing TAMs induce transient vascular leakage via vascular endothelial growth factor A (VEGFA) signaling, thereby facilitating tumor cell intravasation. Interestingly, intravital imaging also showed that transendothelial migration preferentially occurred in regions containing a TMEM, and specifically targeting this site could slow down the spread of cancer cells28. Intravital imaging has also been used recently to compare the motility of two immune effector cell types, natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs)29. Here, the authors showed that while NK cells form short and fast contacts with tumor cells independent of calcium signaling, CTLs form long-lasting contacts with tumor cells that require calcium influx into CTLs. Interestingly, both cell types were shown to require calcium influx for efficient killing29, thus intravital imaging has provided new insights into the cell-type-specific interactions between tumor cells and the immune system which are needed for specific and efficient killing of tumor cells to occur and which cannot be mimicked faithfully in vitro.\n\nOn a similar note, using intravital imaging, Moalli et al. characterized a potential pathway through which tumor-derived antigen is locally processed and presented to generate a systemic immune response30. Injection of B16.F10 melanoma cells, transfected with tdTomato as a surrogate tumor-derived antigen, into the mouse footpad led to a robust immune response after 30 days measured by high titers of anti-B16.F10-tdTomato IgG30. Imaging of tumor-draining lymph nodes showed that tdTomato was localized in macrophages in the lymphatics and also in follicular dendritic cells (FDCs). Using mouse models of macrophage, FDC, or B-cell deficiency, the authors suggest that tdTomato is taken up by macrophages and subsequently localizes with FDCs, which are then scanned by B-cells, generating a systemic immune response30. Similarly, intravital imaging of mCherry-expressing mammary tumors was used to characterize antigen presentation at the tumor site31. Here, the authors showed that a subset of myeloid cells function as tumor dendritic cells by taking up and presenting tumor-derived antigen to CTLs. Interestingly, it was shown that CTLs engage with the antigen-presenting cells and subsequently become arrested in this engagement, potentially inhibiting cytolytic effects and tumor rejection. The authors suggest that tumor immunotherapy may help release the CTL blockade following their initial attraction and clustering with antigen-presenting cells at the tumor site31. Similarly, Boissonas et al. showed by intravital imaging that infiltrating T-lymphocytes can become trapped with tumor dendritic cells following chemotherapy, suggesting that tumor dendritic cells can function as a sink that retains the T-cell-driven anti-tumor immune response32. These findings enhance our understanding of the immune response to cancer development both at primary and metastatic sites, thus intravital imaging may help pave the way to the development of new presentation-derived therapeutics. For example, intravital imaging in the MMTV-PyMT mouse model of breast cancer enabled the identification of a type of macrophage-dendritic cell (M-DC)33. Here, the authors used fluorescent dextran to label M-DCs and revealed that the depletion of M-DCs decreases tumor growth and metastasis and could therefore present a promising future target33. Similarly, recent intravital imaging of CTL intratumoral migration in vivo revealed that treatment with anti-CD137 mAb prolongs the interaction between CTLs and cancer cells, thereby improving the antitumor CTL activity34. This is in parallel with recent improvements in anti-PD1 immunotherapy to educate the immune system to recognize and kill melanoma cells35 and may represent a promising approach for the improvement of immune-based therapeutics in multiple cancer types.\n\nSeveral fluorescent reporter systems to specifically label and track immune cells within native tissues have been developed recently. One such example is the Catchup mouse model36, wherein a bicistronic cassette of Cre and tdTomato was engineered to be expressed under the neutrophil-specific locus Ly6G. Using this tool, the authors tracked neutrophil migration and studied neutrophil-specific gene functions in vivo. Similarly, a transgenic mouse with ubiquitous expression of Kikume, a green fluorescent protein (GFP) that can be irreversibly photo-converted to red via violet light excitation, was used to characterize dendritic cell37 and neutrophil migration38 in vivo (Figure 1B, single-cell tracking of neutrophils38). For example, upon bacterial infection, neutrophils were recruited to the mouse ear and were photo-converted. Further intravital imaging allowed the authors to track the photo-converted cells and to identify and map new patterns of neutrophil migration and homing into the lymph nodes38. Another example is the MacGreen transgenic reporter mouse in which c-fms-driven GFP expression is targeted to macrophages, trophoblasts, and granulocytes39. Using this reporter, Pai et al. could track circulating leukocytes during cerebral malaria pathogenesis and identified plasmodium-specific CD8+ T lymphocytes as important regulators of this disease40. Crossing these reporter animal models with mouse models of cancer may provide much-needed insights into how cancer cells manipulate and interact with the host immune system to promote tumor progression. For example, intravital imaging may allow us to identify the migration routes of immune cells during cancer progression and treatment or to characterize tumor immune subtypes41,42 in a dynamic manner to better inform on immunotherapies in cancer, a feature of cancer progression that cannot be modeled or assessed accurately in vitro.\n\n\nCancer-fibroblast interactions facilitate tumor progression\n\nFibroblasts are also frequently recruited to tumor sites, and intravital imaging has recently been used to show that stromal fibroblasts can regulate overall tumor response to chemotherapy. For example, intravital imaging of a xenograft model of melanoma through an optical window demonstrated paradoxical activation of cancer-associated fibroblasts (CAFs) at the primary site upon treatment with Braf inhibitors, thereby leading to increased stiffening of the ECM compartment43. Here, the authors show that the stiffened ECM in turn activated adhesion-mediated signaling pathways, such as focal adhesion kinase (FAK), to promote cancer cell survival and so decrease anti-Braf treatment efficacy43. Interestingly, impairing cellular adhesion tension through FAK inhibition reduced the safe haven provided by the ECM and resulted in a significantly enhanced response to chemotherapy43.\n\nSimilarly, Lee et al. used in vivo whole-body bioluminescence imaging to investigate the mechanisms of resistance to anti-insulin-like growth factor (anti-IGF) treatment with cixutumumab44, which recently showed limited efficacy in clinical trials45,46. Here, the authors demonstrated that upon treatment with cixutumumab, cancer cells underwent a protective reprogramming characterized by increased production of IGF, which in turn led to the recruitment and activation of host fibroblasts to the primary site through IGF-2R-dependent paracrine signaling. Activated fibroblasts were then shown to secrete pro-angiogenic factors and to potentiate metastasis and disease relapse44. Their findings also correlated with changes in the tumor microenvironment of patients undergoing anti-IGF-based therapies44, suggesting that preventing fibroblast activation can impair extrinsic mechanisms of chemoresistance and in turn improve cixutumumab efficacy.\n\nIntravital imaging has also been used to implicate stromal fibroblasts in cancer cell dormancy, an important cause of disease relapse, which remains poorly understood47. In a recent study, Lawson et al. developed an approach for longitudinal intravital imaging of myeloma cells that had lodged within dense bone tissue48. Using a dye-dilution system, the authors were able to label and track slowly proliferating, dormant cells over time and showed that these cells preferentially lodge in direct contact with the endosteal bone surface and interact with host bone cells to establish a protective niche for persistence of metastasis (see Figure 1C showing dormant cells [red] lodged at the surface of the bone [white] and proliferating cells [green]48). Importantly, this study demonstrated that cell dormancy is a reversible state, which can be turned “off” and “on” by signals emanating from host osteoblasts and osteoclasts. The authors suggest that using bone-active drugs to either prevent the reactivation of dormant cells or take cells out from dormancy before treating them with chemotherapy could improve outcome and reduce disease relapse. Their findings present new opportunities for the treatment of metastatic cancers with a bone tropism such as breast, prostate, or kidney cancer48.\n\nTumor cells have also been shown to co-opt fibroblasts to promote cancer spread, and recent in situ imaging of secondary organ colonization has been used to reveal how host fibroblasts are implicated in the preparation of the metastatic niche49. In an orthotopic model of breast cancer, dual monitoring of metastatic initiating breast cancer cells and lung fibroblasts recently revealed that reciprocal interactions between the two cell types are required for successful establishment of lung metastases. Here, the authors demonstrate that a biphasic intercellular crosstalk progressively modifies both compartments. Briefly, activation of a fibrotic program in the fibroblast population led to the initiation of a mesenchymal-to-epithelial transition within the cancer cell compartment, promoting the onset of metastasis49. Taken together, these studies suggest that preventing fibroblast activation and reprogramming driven by tumor cells during cancer progression may help improve sensitivity to chemotherapy and impair cancer dissemination. Intravital imaging of secondary sites prone to metastasis is required to directly visualize the stages of metastatic colonization; however, long-term and deep imaging of metastatic organs remains technically challenging. Optical imaging windows now enable longitudinal and deep imaging of events occurring at both primary sites and secondary organs such as the liver, spleen, kidney, and pancreas50,51. For instance, monitoring the early events promoting the formation of the liver metastatic niche was achieved using optical imaging windows52. Interestingly, this study demonstrated that single extravasated cells are initially highly motile and proliferative and form pre-micrometastases within the liver before merging into micrometastases with reduced migration. Importantly, impairing early tumor cell migration significantly reduced the metastatic burden52. Similarly, the formation of the liver metastatic microenvironment and response to chemotherapy has been studied using longitudinal scanning multiphoton microscopy in an intrasplenic model of colorectal cancer50 and informed on the response of metastatic cells to chemotherapy.\n\nOne limitation to longitudinal intravital imaging is the movement of live tissue, such as heart or lung, which can perturb high-resolution intravital imaging, and current research focuses on motion compensation systems to correct for tissue movement53–55. Additionally, the probability of imaging an invasive or metastatic event can be considered very low; however, optogenetic intravital imaging can be used to specifically activate or inhibit signaling pathways and subsequently trigger such an event.\n\n\nIntercellular crosstalk is supported by extracellular vesicles\n\nReciprocal interactions between cancer and stromal cells require an exchange of information, for example, in the form of physical cell-cell interaction or secretion of protein ligands or extracellular vesicles (EVs). Direct visualization of signal transmission between cancer cells and the host environment has long been technically challenging; however, the recent development of new intravital imaging tools has helped elucidate the mechanisms which enable intercellular exchange of information within both primary and metastatic sites. Recent studies showed that cancer cells can manipulate host stromal cells through the local and systemic transfer of exosomes and other EVs56–58. For instance, live imaging of a chicken embryo model of fibrosarcoma indicated that exosomes promote cancer cell-ECM adhesion assembly and are required for persistent cell movement59. In addition, elegant in situ studies demonstrated that exosomes secreted from primary cancer cells localize at secondary metastatic sites, fuse with host cells, and can “educate” secondary organs through the activation of fibrotic and inflammatory programs56,57. In line with this study, direct intravital imaging of exosome secretion and uptake by different cell populations60,61 has recently been achieved. Here, Zomer et al. developed a Cre-loxP-based system whereby cells that take up Cre(+) EVs exhibit a change in color (see Figure 1D showing Cre[+] cells [blue], reporter cells that incorporated Cre[+] EVs [green], and reporter cells that did not60). With this approach, the authors demonstrated that EVs are locally and systemically transferred from aggressive cancer cells to less malignant cancer cells within the same mouse to coordinate a whole-body systemic metastatic program in vivo and to enhance the overall metastatic ability of the tumor60,61. Recent findings also demonstrate that mitochondrial DNA (mtDNA) can be transferred from host stromal cells to cancer cells. Here, the authors genetically manipulated melanoma and breast cancer cells to lack mtDNA and showed in situ that they can acquire host mtDNA to restore mitochondrial functions such as bioenergetic dynamics and respiration. In addition, incorporation of host mtDNA by cancer cells led to increased tumor growth and metastatic spread, demonstrating that tumor cells can take up material from the host to drive cancer aggressiveness62,63. Interestingly, Osswald et al. used in vivo multiphoton scanning of astrocytomas over a year to study a novel mechanism of intercellular connection64, wherein tumor cells in astrocytomas use ultra-long protrusions (>500 μm in length) to form a network of multi-cellular communication. The authors show that this network supports tumor cell invasion and proliferation and protects cancer cells against radiotherapies64. Future developments in intravital imaging to directly visualize mechanisms of cell-cell interaction may help us understand how cancer cells and host tissues interact and together drive cancer aggressiveness, and in the future may help us to uncouple this support mechanism.\n\n\nImaging biomechanical properties of the extracellular matrix\n\nOver the last decade, intensive work has shown that ECM abundance and features, such as stiffness, topography, and porosity, contribute to tumor cell proliferation, invasion, and chemoresistance65–72. Multiphoton imaging technologies, such as second harmonic generation (SHG) imaging73, have been used to study the modulation of ECM characteristics during cancer progression and have recently identified new regulators of biomechanical tissue properties (see Figure 1E showing collagen deposition in in vitro 3D organotypic fibroblast-collagen matrices upon induction of P-Rex, a guanine exchange factor for Rac174). For instance, the actin cytoskeleton and a number of its regulatory proteins, such as the family of Rho GTPases, are known to govern the remodeling of the ECM69,75–77, and live SHG imaging has provided new insights into the tight regulation of Rho GTPases in vivo. In collaboration with Samuel and colleagues, we helped identify the intracellular signaling protein 14-3-3 ζ as a novel negative regulator of Rho-kinase-driven ECM stiffening in wound healing78. Here, live monitoring of stromal fibroblasts coupled with SHG imaging of collagen fibers in a 3D in vitro environment was used to show that loss of 14-3-3 ζ in dermal fibroblasts impairs their ability to remodel collagen and to stiffen the ECM, and such effects were also seen in ex vivo animal samples78. Interestingly, this study also showed in squamous cell carcinoma that 14-3-3 ζ is downregulated while collagen deposition is increased, suggesting that ROCK signaling is no longer restrained and controlled in vivo in this disease69,78,79. These findings suggest new therapeutic options for re-purposing 14-3-3 ζ-based treatments to facilitate uniformed and controlled wound healing78, which in future can also be used in the context of cancer to re-establish normal tissue homeostasis of solid tumors69,80,81.\n\nThe hypoxic tumor environment was also recently identified as a novel regulator of the ECM ultrastructure and stiffness at both primary and metastatic sites82,83. For example, hypoxia was shown to inhibit prolyl hydroxylase domain protein 2 (PHD2) in CAFs in vitro and in vivo, leading to a reversion of CAF activation and a decrease in their ability to remodel and stiffen the ECM82. In addition, proteins secreted from hypoxic tumors were demonstrated to prepare and activate the ECM of future metastatic sites. One such example is the enzyme lysyl oxidase (LOX), which is upregulated in bone tropic breast cancer cells83. In vivo, injection of LOX resulted in the formation of osteolytic lesions and in turn provided circulating breast cancer cells with a focal pre-metastatic niche for bone colonization, whereas LOX targeting with a blocking antibody partially reverted this phenotype and decreased tumor burden, as visualized by whole-body in vivo imaging83. Intravital SHG imaging has also been used to directly monitor changes of the ECM structure and content during cancer progression in live settings. For example, Walsh et al. performed intravital SHG imaging of breast cancer xenografts to study the effects of trastuzumab treatment on the ECM84. Interestingly, their study demonstrated that trastuzumab induces changes in collagen density and alignment and thereby identified a non-cellular response to drug treatment84. In addition, longitudinal SHG imaging of orthotopic breast xenografts was performed at the tumor margin to identify the key components of the tumor microenvironment that modulates cell invasion85. Here, the authors used dual imaging of single cells and collagen fibers and showed that slow-locomotion breast cancer cells form invadopodia to remodel and stiffen the collagen matrix while they move through the surrounding tissue. Further use of intravital SHG imaging will provide important insights into the role of the ECM and its manipulation during tumor progression.\n\nRapidly developing intravital technologies and tools have provided unprecedented insights into cancer complexity and have also opened up promising avenues for pre-clinical cancer research. For example, SHG imaging has recently been translated to study the properties of the ECM in biopsy samples of cancer patients. For instance, in the context of pancreatic cancer, we recently used SHG imaging to analyze the properties of the ECM in a human pancreatic tissue microarray (>80 samples) and detected a positive correlation between fibrillar collagen abundance, tumor stage, lymph node spread, and vascular invasion, suggesting that fine-tuned targeting of collagen crosslinking may be a valid approach in this disease86. In line with this study, biophysical analysis of collagen remodeling was conducted in human breast cancer biopsies (>20 samples) and revealed a progressive increase of collagen reorganization and orientation as invasive cancer lesions develop87. Similarly, in a cohort of matched human breast tissues with areas of high and low mammographic density from the same patient (>15 samples), we analyzed ECM organization by gray-level co-occurrence matrix (GLCM), an image texture-based approach used to quantify the organization of collagen fiber networks88. In this study, Britt and colleagues demonstrated that increased deposition and crosslinking of collagen fibers correlates with a high risk of developing breast cancer88. Taken together, these studies along with our knowledge from intravital animal models indicate that upon acquisition of human biopsies, characterization of ECM abundance and crosslinking using medium-throughput SHG imaging can potentially be used as a biomarker to predict patient prognosis and tumor stage. Moreover, the development of new probes to directly image distinct components of the ECM such as hyaluronan3,89, elastin90, or fibronectin91 is likely to facilitate our understanding of the role of the ECM in cancer development and may help us improve therapies targeting specific components of the ECM.\n\n\nNew tools for live, intravital, and in situ imaging in translational cancer research\n\nFluorescence-based biosensors are emerging as a reliable tool to dissect mechanisms of drug target activity and chemoresistance. In a large panel of MEK inhibitor (MEKi)-resistant cell lines, live imaging of extracellular signal-regulated kinase (Erk) and S6K fluorescent biosensors was used to interrogate the molecular bases of resistance to MEKi in Kras-mutant and Braf-mutant cancer cell lines in vitro. Here, the authors showed that upon treatment with MEKi, Erk and PI3K pathways can maintain mTORC1 activity and in turn promote cell growth, thereby leading to resistance to treatment92. Furthermore, fluorescently tagged drugs were used to decipher mechanisms of drug resistance. For instance, a fluorescent analog of eribulin was designed to monitor in vivo drug pharmacokinetics. Here, the authors engineered a portion of tumor cells to express multidrug-resistance 1 (MDR1)-mApple fusion protein, and dual imaging of eribulin and MDR1-mApple demonstrated that resistance to eribulin was directly correlated with vascular architecture and MDR1-mediated drug efflux. This study also demonstrated that inhibition of MDR1 reversed the multidrug-resistant phenotype and improved eribulin efficacy93 (Figure 1F showing fluorescent eribulin [green] diffusing into tumor tissue and being incorporated only by cancer cells not expressing MDR1-mApple fusion protein at 1 and 2 hours following treatment with eribulin93).\n\nTaking these approaches further, for the assessment of whole-tissue or whole-body protein activity, transgenic mice have been generated with ubiquitous or Cre-inducible expression of reporters of the activity of Erk, PKA, Rac1, cAMP, or cell cycle progression94–99 (Figure 1G, map of Rac activity where red and yellow mark low Rac activity and blue and green highlight high Rac activity96). Crossing these biosensor mice with animal models of cancer allows us to dissect the spatiotemporal mechanisms of the molecular events driving cancer in native tissues. For example, Kumagai et al. recently crossed their Erk biosensor mouse with a mouse model of mammary tumor formation and described a stable heterogeneous pattern of Erk activity100. Interestingly, the authors showed that cells with low Erk activity were more successful in forming tumor spheres in vitro and expressed high markers of mammary cancer stem cells compared to cells with high Erk activity100. These findings indicate that tumor heterogeneity in Erk activity may be beneficial to establish cancer stem cells with low Erk activity for self-renewal, whereas high Erk activity supports rapid growth and expansion100. Similarly, Erk pulsing and propagation dynamics emanating from the skin and hair follicle have also recently been demonstrated in vivo101 and have significant implications for future research in melanoma102.\n\nAnother recent example of biosensor mouse imaging in cancer is our E-cadherin-GFP mouse, which enabled us to perform photobleaching experiments in vivo to monitor cell-cell junction dynamics (Figure 1G, E-cadherin-GFP expression in cell-cell junctions of the lactating mammary gland103). Here, we used fluorescence recovery after photobleach (FRAP) and fluorescence loss in photobleach (FLIP) imaging along with kymograph analysis to quantify E-cadherin mobility of healthy and diseased tissue. Using this approach, we could correlate high E-cadherin mobility in the cell membrane with a decrease in junction strength and integrity and an increase in cell invasiveness103. Crossing this mouse with a genetically engineered model of pancreatic cancer allowed us to recapitulate the full spectrum of pancreatic cancer progression and to dissect the genetic stages of early cancer dissolution. This mouse was also used as a tool for drug discovery by testing new anti-invasive agents, such as dasatinib, in pancreatic cancer, where we demonstrated that dasatinib is able to strengthen cell-cell junction integrity in line with decreased invasiveness in this setting103. This is in line with current clinical assessment in patients and its known effectiveness in the KPC pancreatic cancer mouse model104.\n\nMouse models with ubiquitous expression of molecular reporters can now be used to monitor events in a large range of organs and diseases; however, due to light scattering within tissues, the imaging depth with current two-photon lasers is limited to several-hundred μm, limiting intravital imaging of large organs and tumors. Current multiphoton setups include three-photon lasers in combination with optical parametric oscillators (OPOs) that increase the excitation wavelength, as red and near-infrared light can penetrate deeper into tissue and reduces tissue scattering105,106. This requires the use of fluorescent proteins that can be excited with near-infrared wavelengths, such as iRFP or iRFP variants, which have been recently described107,108. Furthermore, computational models, such as adaptive optical correction, have been used to overcome light scattering and to increase imaging depth109. Another imaging technique, called photoacoustic tomography (PAT), makes use of the phenomenon that photon absorption forms pressure waves that can be detected. Although PAT increases imaging depth, it strongly reduces image resolution, which can be partially overcome by the use of near-infrared fluorescent proteins110,111. Furthermore, current proceedings to reduce the distal optics and mechanical components of intravital microscopes112–114 should move the field towards intravital endoscopy of cancer biology115.\n\nLastly, optogenetics, which rely on the inherent properties of light to observe and accurately control complex biochemical and signaling events116, is emerging as another promising tool to interrogate cancer dynamics. Optogenetics allows researchers to manipulate gene expression or the activity of cellular signaling pathways and has recently enabled photocontrol of proteins with subcellular resolution. These approaches have been used to modify protein conformation117, stimulate DNA binding118, control enzymatic119 or receptor tyrosine kinase activity120, induce protein localization121, manipulate protein-protein interactions122, and study signaling pathway events123,124. For instance, optogenetics was used to demonstrate that localized Rac1 activity is sufficient to produce precisely regulated cell protrusion events to control directed cell motility in vivo125–128. Most importantly, optogenetics may enable us to modulate cancer-related signaling pathways in a defined spatiotemporal manner and allow us to follow the fate of de-regulated cells in their in situ environment. In summary, intravital imaging has demonstrated its capacity to allow us to explore the ancillary mechanisms supporting cancer progression and provide novel routes for the development of future therapeutics to target and impair these support mechanisms.\n\n\nAbbreviations\n\nCAF, cancer-associated fibroblast; CAM, chorioallantoic membrane; CTL, cytotoxic T-lymphocyte; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; EV, extracellular vesicle; FAK, focal adhesion kinase; FDC, follicular dendritic cell; FLIP, fluorescence loss in photobleaching; FRAP, fluorescence recovery after photobleaching; GLCM: gray-level co-occurrence matrix; IGF, insulin-like growth factor; LOX, lysyl oxidase; M-DC, macrophage-dendritic cell; MDR1, multi-drug resistance 1; MEKi, MEK inhibitor; mtDNA, mitochondrial DNA; NK cell, natural killer cell; OPO, optical parametric oscillator; PAT, photoacoustic tomography; PHD2, prolyl hydroxylase domain protein 2; QD, quantum dot; SHG, second harmonic generation; TAM, tumor-associated macrophage; TMEM, tumor microenvironment of metastasis; VEGFA, vascular endothelial growth factor A.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nClaire Vennin, David Herrmann, Morghan C. Lucas, and Paul Timpson were funded by an NHMRC project grant, an ARC Future and a Len Ainsworth Pancreatic Cancer Fellowship, the Cancer Council NSW, and a Tour de Cure grant.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors wish to thank Dr Sean C. Warren, Dr Andrew Burgess, and Dr David R. Croucher for critical reading of the manuscript.\n\n\nReferences\n\nZheng Y, Chen J, Craven M, et al.: In vitro microvessels for the study of angiogenesis and thrombosis. Proc Natl Acad Sci U S A. 2012; 109(24): 9342–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFernandez CE, Yen RW, Perez SM, et al.: Human Vascular Microphysiological System for in vitro Drug Screening. Sci Rep. 2016; 6: 21579. 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Publisher Full Text\n\nPapadopoulos IN, Farahi S, Moser C, et al.: High-resolution, lensless endoscope based on digital scanning through a multimode optical fiber. Biomed Opt Express. 2013; 4(2): 260–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSherlock B, Warren S, Stone J, et al.: Fibre-coupled multiphoton microscope with adaptive motion compensation. Biomed Opt Express. 2015; 6(5): 1876–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOh G, Yoo SW, Jung Y, et al.: Intravital imaging of mouse colonic adenoma using MMP-based molecular probes with multi-channel fluorescence endoscopy. Biomed Opt Express. 2014; 5(5): 1677–89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeisseroth K: Optogenetics. Nat Methods. 2011; 8(1): 26–9. PubMed Abstract | Publisher Full Text\n\nGasser C, Taiber S, Yeh CM, et al.: Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase. Proc Natl Acad Sci U S A. 2014; 111(24): 8803–8. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nStrickland D, Moffat K, Sosnick TR: Light-activated DNA binding in a designed allosteric protein. Proc Natl Acad Sci U S A. 2008; 105(31): 10709–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYi JJ, Wang H, Vilela M, et al.: Manipulation of endogenous kinase activity in living cells using photoswitchable inhibitory peptides. ACS Synth Biol. 2014; 3(11): 788–95. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGrusch M, Schelch K, Riedler R, et al.: Spatio-temporally precise activation of engineered receptor tyrosine kinases by light. EMBO J. 2014; 33(15): 1713–26. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nZhou XX, Chung HK, Lam AJ, et al.: Optical control of protein activity by fluorescent protein domains. Science. 2012; 338(6108): 810–4. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBaarlink C, Wang H, Grosse R: Nuclear actin network assembly by formins regulates the SRF coactivator MAL. Science. 2013; 340(6134): 864–7. PubMed Abstract | Publisher Full Text\n\nToettcher JE, Weiner OD, Lim WA: Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell. 2013; 155(6): 1422–34. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKatsura Y, Kubota H, Kunida K, et al.: An optogenetic system for interrogating the temporal dynamics of Akt. Sci Rep. 2015; 5: 14589. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWang X, He L, Wu YI, et al.: Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nat Cell Biol. 2010; 12(6): 591–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWu YI, Wang X, He L, et al.: Spatiotemporal control of small GTPases with light using the LOV domain. Meth Enzymol. 2011; 497: 393–407. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu YI, Frey D, Lungu OI, et al.: A genetically encoded photoactivatable Rac controls the motility of living cells. Nature. 2009; 461(7260): 104–8. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nO'Neill PR, Gautam N: Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration. Mol Biol Cell. 2014; 25(15): 2305–14. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "13861",
"date": "16 May 2016",
"name": "Jacco van Rheenen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13862",
"date": "16 May 2016",
"name": "Michiyuki Matsuda",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-892
|
https://f1000research.com/articles/5-888/v1
|
13 May 16
|
{
"type": "Software Tool Article",
"title": "A cloud-based learning environment for comparing RNA-seq aligners",
"authors": [
"Elizabeth Baskin",
"Peter DeFord",
"Allison F. Dennis",
"Ian Misner",
"Frederick J. Tan",
"Ben Busby",
"Elizabeth Baskin",
"Peter DeFord",
"Allison F. Dennis",
"Ian Misner",
"Frederick J. Tan"
],
"abstract": "The rapid rise of high-throughput, data intensive experimental techniques has thrust many biologists into the role of data analyst – a role many biologists feel ill equipped to fill. Novices often struggle to find the resources and expertise they need to analyze their experimental results in a wet-lab environment. To fill this need, we developed an educational resource as part of a National Center for Biotechnology Information (NCBI) hackathon. Using RNA-seq as a model, our tutorial guides new users through the steps of data analysis, while placing an emphasis on understanding the motivation behind choices made in the process. To advance the goal of providing a deeper understanding of the analysis process, we developed a new tool, bamDiff. bamDiff allows users to compare the performance of multiple RNA-seq aligners, allowing users to select the most appropriate aligner for the data in question and experimental end-goal. Our tutorial is accessible via a GitHub wiki, with associated data and software provided on an Amazon Machine Image (AMI), which can be completed at no cost to the user through the Amazon Educate Program. Following the hackathon, our tutorial was integrated into the October 2015 offering of NCBI NOW (Next Generation Sequencing (NGS) Online Workshop) a free online experience targeting individuals new to NGS analysis.",
"keywords": [
"RNA-seq",
"SAM/BAM alignments",
"education",
"cloud",
"hackathon",
"pipeline",
"workflow",
"alignment"
],
"content": "Introduction\n\nWith the rise of RNA-seq for exploring biological hypotheses has come an increase in the number of algorithms for aligning RNA-sequences to the genome. The burden of selecting and properly using these algorithms often falls on biologists. However, many biologists do not have training or experience in the skills needed to select the proper tools. To provide an introduction for this audience, the Educational Environment Team sought to develop an interactive learning environment where students with a novice’s background in Unix could follow a series of alignment pipelines step-by-step.\n\nThe team identified two major goals that could be undertaken within the scope of the hackathon. The first was to produce a user-friendly tutorial that would walk novice computational biologists through the process of aligning RNA-seq data to the genome. The second was to supplement the tutorial with novel methods to compare the results of different RNA-alignment mappers.\n\nImplementing this tutorial on an Amazon Machine Image (AMI) -- a virtual server in the cloud, pre-programmed with all the necessary packages -- allows students to initially bypass the intimidating task of installing software and dependencies, and immediately start performing alignments using a panel of different algorithms. All tasks in the tutorial and the AMI are designed to fall within the $35 per student provided for free through the Amazon Educate Program (http://aws.amazon.com/education/awseducate). By allowing students to run these alignments in succession, we hope to naturally showcase how these aligners vary in their outputs. Here we introduce both a read-based and position-based approach for identifying and evaluating regions of differential performance across the genome.\n\n\nMethods\n\nThe team was composed of five members ranging in experience from graduate student to research faculty member. Each member of the team shared the experience of interfacing with non-computational biologists during their daily work and sought to provide a welcoming introduction to mapping tools. All members contributed to the goals of the team, with each selecting tasks to tackle according to their strengths.\n\nAs part of the tutorial, two team members dedicated their time to developing novel methods for comparing the results of RNA-alignment. After each of the town-hall style meetings conducted with the larger hackathon group, we received recommendations from members of other teams for software packages to help streamline the proposed analysis.\n\n\nResults\n\nThe tutorial was constructed as a GitHub Pages wiki -- each new wiki page represents a new step in the workflow1. We guide students through registration with the Amazon Educate Program, obtainment of data, alignment, and comparison of the performance of four different aligners. The comparison of aligners is achieved with our own custom written program, bamDiff, in concert with the R (v3.2.1) packages edgeR (v3.10) and csaw (v1.2.1).\n\nThe example dataset is sample NA12878 from the Genome in a Bottle Consortium, an extensively curated human-standard2. To give a flavor for the alignment workflow without burdening the learner with expensive computational requirements, we limited the genome for alignment to chromosome 20 of the latest human genome reference from RefSeq, GRCh38 (GCF_000001405.30). For the tutorial, we describe the use of four popular aligners: BWA v0.7.12-r10393, HISAT v0.1.6-beta4, STAR v2.4.0j5, and BLASTmapper (in preparation). For each, we provide the commands needed to construct an index and align the given data, outlining the expected screen output, files to be created, and time required for each task.\n\nThe R package csaw provides an elegant framework for identifying regions of differential expression between RNA-seq experiments, and in this case was extended to identifying regions of differential alignment between mappers6. To implement read counting, we used csaw to bin reads into 1KB windows, then filtered windows by count size7. Only bins with greater than 10 reads were kept. Windows were then filtered by exons, and only windows/bins containing exons were kept for further analysis (see Figure 1c). edgeR was used to identify windows/regions/bins with significant differences between the four mappers (see Figure 1d)8.\n\n(a) Raw RNA sequencing reads undergo quality control checks and filtering before (b) being aligned using several different aligners. (c) csaw bins the reads and filters for bins based on read counts and overlap with exons. (d) edgeR identifies regions of where the aligners show differential mapping in the csaw filtered regions. (e) from the edgeR identified regions, bamDiff checks where those reads are being mapped in the other BAM files.\n\nIn order to systematically compare the performance of the various aligners, we wrote a new program called bamDiff. This Python script takes the output from csaw and edgeR as a CSV file as well as the outputs from each aligner in BAM format. bamDiff will report simple summaries for each BAM file, such as the total number of reads, overall number of alignments, proportion of reported reads that were unmapped by the aligner, as well as proportion of aligned reads that were mapped only once (uniquely).\n\nThe real strength of bamDiff comes in its ability to go beyond simple summarization to direct comparisons between BAM files. Internally bamDiff uses SAMtools9 to rapidly extract only the reads mapping to a region of interest identified by csaw from one BAM file. These reads are then checked against the other BAM files to see whether they are mapped at all, and if so, whether they are mapped to the same region in the genome as in the first BAM file (see Figure 1e). If they map to a conflicting region outside the region of interest, bamDiff will report the top ten regions reads are mapping to, by agglomerating reads mapping within 1kb of each other. These results are reported as text tables. Example usage and output can be viewed at the associated page in our tutorial: https://github.com/NCBI-Hackathons/RNA_mapping/wiki/7.-Compare-alignments-with-bamDiff.\n\nFollowing the hackathon, our tutorial was integrated into NCBI NOW (National Center for Biotechnology Information Next Generation Sequencing (NGS) Online Workshop) a free online experience targeting individuals new to NGS analysis. The first offering of the workshop occurred October 13–23, 2015. Our tutorial was offered as a “take-home” exercise following the sixth lecture, which focused on the analysis of RNA-seq data.\n\n\nConclusions and next steps\n\nWe achieved two distinct goals during the hackathon: first, the development of a novel method for comparing the results of RNA-alignment and second, the creation of a tutorial to guide users through not only undertaking, but also understanding, RNA-seq alignment. Notably, we do not over-simplify the work of data analysis by simply selecting a single aligner for use in our tutorial. Instead, by encouraging the comparison of many different aligners we draw attention to the decision-making process intrinsic to any bioinformatic work. For our user, this is the selection of an RNA-seq aligner from many frustratingly similar options.\n\nHowever, both the novel software bamDiff and the tutorial can be further improved.\n\nCurrently, bamDiff is best suited to analyzing a small region of the genome/BAM file, rather than undertaking the entire genome/BAM file. Additionally, a graphical summary would make outputs easier to understand.\n\nIn the tutorial we assume prior knowledge of the contents, purpose, and format of FASTQ, SAM, and BAM files as we assumed that our tutorial would be used in conjunction with a class or workshop that covers these introductory topics, such as NCBI NOW. The tutorial could be expanded in a number of ways, such as generalizing to pair-end reads or alignments guided by an annotation. Finally, there may be room for improvements in usability, such as streamlining use on platforms such as Google Cloud, Microsoft Azure, or iPlant/CyVerse.\n\n\nSoftware availability\n\nLatest source code: https://github.com/NCBI-Hackathons/RNA_mapping.\n\nSource code as at the time of publication: http://dx.doi.org/10.5281/zenodo.5087110\n\nLicense: CC0 1.0 Universal",
"appendix": "Author contributions\n\n\n\nAll of the authors participated in designing the study, carrying out the research, and preparing 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 work on this project by Ben Busby was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Library of Medicine (NLM), and NCBI. Elizabeth Baskin was supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases (Z01-AR041198). Peter DeFord was supported by NIH Training Grant GM007231.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors thank Lisa Federer, NIH Library Writing Center, for manuscript editing assistance.\n\n\nReferences\n\nRNA Mapping Team: RNA Mapping Github [Internet]. RNA Mapping Github. [cited 2016 Feb 16]. Reference Source\n\nNational Center for Biotechnology Information: Bioproject [Internet]. Homo Sapiens ID 236780. 2014. [cited 2016 Feb 16]. Reference Source\n\nLi H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14): 1754–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim D, Langmead B, Salzberg SL: HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015; 12(4): 357–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDobin A, Davis CA, Schlesinger F, et al.: STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29(1): 15–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLun AT, Smyth GK: csaw: a Bioconductor package for differential binding analysis of ChIP-seq data using sliding windows. Nucleic Acids Res. 2016; 44(5): e45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLun A, Smyth GK: csaw user manual [Internet]. 2016. [cited 2016 May 2]. Reference Source\n\nRobinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26(1): 139–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeFord P, Tan F, Misner I, et al.: RNA_mapping: RNA_mapping. Zenodo. 2016. Publisher Full Text"
}
|
[
{
"id": "15576",
"date": "05 Sep 2016",
"name": "Malachi Griffith",
"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 the results of an educational effort in which a hackathon event was used to develop an interactive tutorial to help biologists learn a fundamental NGS analysis skill. Specifically, that of selecting an appropriate read aligner, performing alignments, and evaluating the outcome. Overall the tutorial is organized, and the accompanying paper is well written.\nMajor points:\nThe primary goal of this work is commendable. However having reviewed the paper and tutorial, I was surprised by the lack of discussion/ interpretation of the results. Choosing an appropriate RNA-seq read aligner and evaluating the outcome can indeed be a challenge to those new to the field. The tutorial walks a user through the process of conducting alignments with four possible aligners. Some tools that evaluate the resulting aligners are presented and used during the tutorial. However, the authors offer little interpretation of the results, even for the demonstration data set. What do the results tell us about the quality of each alignment result? What factors might be considered in deciding which is \"best\"? What are the pitfalls for such assessments? How might the results be visualized to assist interpretation?\n\nA secondary goal of creating a tool \"bamDiff\" to assist comparisons between RNA-seq aligners is less well developed. This works seems to be fairly preliminary at this stage, consisting of a single Python script that produces a text summary of a few metrics extracted from RNA-seq BAMs from multiple aligners. Similar to the previous point, additional development would be needed before the results of this tool would be readily useful to most prospective users.\n\nConsiderable resources/ tools for performing quality assessment of BAM files (including RNA-seq alignments) already exist. The authors could provide an overview of these, either in the paper or as an additional section in the tutorial Wiki.\n\nIn the tutorial. The section for each aligner considered (BWA, HISAT, STAR, and blastmapper) should provide a basic description of the aligner, references, link to the aligner documentation, etc.\n\nThe alignment comparisons focus on the number of reads aligned, and how aligners differ in the alignment of particular reads, or reads aligning to particular regions. What other ways might the aligners be different? For example, in their ability to correctly map RNA-seq reads across exon-exon junctions, align reads containing single base sequencing errors or polymorphisms, correctly handle reads containing small insertions or deletions relative to the reference genome, etc.\n\nMinor points:\nPerhaps the abstract should include a URL for the tutorial mentioned in the title.\n\nThe authors have created an AMI to \"allow students to initially bypass the intimidating task of installing software and dependencies\". This is reasonable, but perhaps the installation task could be provided (with detailed instructions) as an optional exercise.\n\nOn a related note, it would be ideal to have detailed documentation on how the AMI (ami-3590de50) was configured (including all dependencies that were installed).\n\nIn addition, this tutorial could include a \"resources/pre-reading\" section that referred the reader to additional helpful materials on RNA-seq sequencing and analysis principles (in addition to the hands on pre-requisites already listed in section 1).\n\nMore details on the example RNA-seq data set used in the hands on exercises would be helpful.\n\nAre there similar efforts for comparison of DNA aligners that could be referenced by this tutorial?\n\nOther RNA-seq educational pieces that cover many topics relevant to new NGS users (with less focus on aligner comparison specifically) could be cited by this paper (e.g. Griffith M et al., www.rnaseq.wiki).",
"responses": []
},
{
"id": "16413",
"date": "26 Sep 2016",
"name": "Timothy I. Shaw",
"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 summarizes two achievements from NCBI’s hackathon of 2015. First is a tutorial to introduce RNAseq mapping. Second is bamDiff, a program for comparing different RNA-seq aligner mapping. This reviewer believes the current state of the tutorial is on the thin side and could benefit from additional expansion. The functionality of bamDiff program is intriguing but based on the current state of the program the reviewer feels the program should be expanded to incorporate other QC metrics. Overall, this work makes great stride for guiding biologist to their first hands-on-experience on NGS.\n\nMajor points:\nThe tutorial provides a step by step tutorial from downloading to mapping and some mapping evaluation. The tutorial can be useful to users that find difficulty working on a Unix environment. In the current form, the author introduces basic commands for performing mapping; however, the author should caution and educate the reader that additional vetting of the raw RNAseq mapping is necessary. While mapping evaluation is important, that is just one of the many QC metric necessary in RNAseq data that contribute to the decision making. Here is an incomplete list of RNAseq related issues that should be included in the tutorial:\n\nWhether the RNAseq sample require trimming of low quality or adaptor sequences. The different parameter tuning that increases the coverage of these hard to map region (i.e. STAR 2-pass). At what sequencing depth is the RNAseq sample is deep enough for expression analysis, gene fusion detection, splicing detection, and whether additional sequencing is necessary.\n\n(The reviewer recognize that the above comments might not be suitable within the tutorial, but the author should make some attempt to inform the user of these caveats.)\n\nRegarding the bam comparison program, the author might want to automatically include output of coverage Bed files that can be displayed in UCSC genome browser or IGV. In the tutorial, the author included some examples of coverage differences; however, the discussions from the tutorial appear to be incomplete. The author failed to discuss the reason that contributes to these differences in coverage such as the parameters or algorithm design. Another discussion point should be to examine where the same read is being mapped to in different program? Here I present a couple factors that might impact the mapping the author might want to consider:\nGC content Highly repetitive region Paralogous gene bodies such as some ribosome genes and mitochondrial genes or histone genes might have varying coverage in different programs.\n\nWithin the tutorial, the author mentions “is every mapper allowing reads to map to the intronic region? After all, this is an RNA-seq experiment -- there should be minimal intronic genetic material.” While the statement is largely true; however, this reviewer believe the author should also mention that there are different type of RNA-seq library specifically TruSeq Stranded Total RNA prep would include contain intronic reads. Poly-A enriched RNAseq experiment could also contain intronic reads for intron retention events.\n\nSince the author appear to discuss splicing region in the tutorial, a more detailed analysis on how different programs deals with the splicing region could be of tremendous interest to certain readers. The author might also want to consider the impact of RNA-seq library protocols on the variation of splice site mapping.\n\nA number of tools have been developed for assessing RNAseq alignment quality. A review tools compared to bamDiff might make a stronger case for the novelty of the program.\n\nA comprehensive table in the tutorial or within the manuscript summarizing all the advantages and disadvantages of different mapping programs will definitely enhance the manuscript.\n\nMinor points:\nPerhaps include other popular aligner such as Tophat as an option.\n\nWhile it is great that the pipeline being on the amazon cloud allows users to bypss installation. Perhaps the author could include installation procedure or reference to individual software if the user wants to install the program on their private server.\n\nMake sure all program provide sufficient commenting. Most of the shell script lack sufficient commenting for what the program will do and why the author chose those parameters.\n\nSentence that might benefit from rephrasing “With the rise of RNA-seq for exploring biological hypotheses has come an increase in the number of algorithms for aligning RNA-sequences to the genome.” Particularly the choice of “has come” is a bit awkward of a wording.\n\nAnother sentence that might benefit from rephrasing “If they map to a conflicting region outside the region of interest, bamDiff will report the top ten regions reads are mapping to, by agglomerating reads mapping within 1kb of each other.”\n\nFor certain details on RNAseq analysis, the author want to refer the reader to the following paper for additional details on RNAseq analysis. “https://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0881-8”\n\nRegarding the accompanying software bamDiff. The summary statistics merely output summary statistics obtained from other mapping programs. The author might want to consider refining the output to a more presentable format like outputting to an excel file.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-888
|
https://f1000research.com/articles/5-887/v1
|
13 May 16
|
{
"type": "Review",
"title": "Substance use during pregnancy",
"authors": [
"Ariadna Forray"
],
"abstract": "Prenatal substance use is a critical public health concern that is linked with several harmful maternal and fetal consequences. The most frequently used substance in pregnancy is tobacco, followed by alcohol, cannabis and other illicit substances. Unfortunately, polysubstance use in pregnancy is common, as well as psychiatric comorbidity, environmental stressors, and limited and disrupted parental care, all of which can compound deleterious maternal and fetal outcomes. There are few existing treatments for prenatal substance use and these mainly comprise behavioral and psychosocial interventions. Contingency management has been shown to be the most efficacious of these. The purpose of this review is to examine the recent literature on the prenatal use of tobacco, alcohol, cannabis, stimulants, and opioids, including the effects of these on maternal and fetal health and the current therapeutic options.",
"keywords": [
"drug abuse",
"pregancy",
"prenatal substance use"
],
"content": "Introduction\n\nIn the United States, women comprise 40% of those with a lifetime drug use disorder and 26% of those who meet criteria for both an alcohol and drug use disorder during the prior 12 months1. Furthermore, women are at highest risk for developing a substance use disorder during their reproductive years (18–44), especially ages 18–292. This means that women who are pregnant or soon to become pregnant are at increased risk for substance abuse. According to a national survey conducted in the United States in 2012, 5.9% of pregnant women use illicit drugs, 8.5% drink alcohol and 15.9% smoke cigarettes3, resulting in over 380,000 offspring exposed to illicit substances, over 550,000 exposed to alcohol and over one million exposed to tobacco in utero. Similar patterns of use have been observed in Europe4,5 and Australia6. The most commonly used substance in pregnancy is nicotine, followed by alcohol, marijuana and cocaine7,8. However, polysubstance use is as high as 50% in some studies7,9. Recently, there has been an increase in opiate use in pregnancy. Between 2000 and 2009, the United States saw a five-fold increase in opiate use in pregnancy, coincident with an “epidemic” of opiate prescription misuse10–12.\n\nThere is little information available on the extent of substance use, other than tobacco, among pregnant women in low-income and middle-income countries. The overall prevalence of tobacco use in these countries is 2.6%, with some countries having much higher maternal rates– up to 15%13. While data on illicit substance use in pregnancy is lacking for most middle- and low-income countries, according to the World Health Organization, cannabis is the most common illicit drug worldwide, followed by amphetamine-type stimulants and opiates14, and, as such, they are likely to be used by women of reproductive age. The limited data available for Africa is from South Africa, and indicates that between 3.6 and 8.8% of pregnant women use illicit substances and 19.6% use alcohol15. The most commonly used illicit substances in South Africa include methamphetamine and cannabis16. Opiate use has also increased in places like Africa and Asia17, and is likely to become more prevalent in pregnancy.\n\nPrenatal substance use can bring about several deleterious consequences for both mother and baby, as described in detail below. The concern for the impact of substances on the developing fetus can motivate some women to curb their drug and alcohol use during pregnancy18. In the only prospective study on prenatal substance use, 96% of women with heaving drinking, 78% of women with marijuana use, 73% of women with cocaine use, and 32% of cigarette smokers succeeded in achieving abstinence during pregnancy9. Offsetting the reduction in pregnancy-related use is the dramatic rise in substance use from 6 to 12 months postpartum9. The study showed relapse in 58% of abstinent smokers, 51% of abstinent women who used alcohol, 41% of abstinent women who used marijuana and 27% of abstinent women who used cocaine in the 3 months following delivery9. Thus, while the levels of abstinence in pregnancy may be high, the impact of this is diminished due to the high rates of relapse postpartum. Unfortunately, maternal relapse happens at a time of high childcare needs and when infant development is dependent on maternal bonding. It is also important to note that this was a study conducted in the United States and that the levels of abstinence may not be equivalent in other countries, especially middle- and low-income countries where women may encounter significant socioeconomic stressors, low levels of education, and limited available treatments for substance use.\n\nAs evidenced by these data, substance use in pregnancy is still a critical public health concern. The purpose of this review is to provide a brief overview of the pregnancy outcomes, neonatal and long-term developmental consequences of prenatal substance use, and current available treatments for pregnant women.\n\n\nAdverse effects of substance use in pregnancy\n\nHeavy alcohol use in pregnancy has been associated with a range of negative birth outcomes, including increased risks of miscarriage19, stillbirth and infant mortality20,21, congenital anomalies22, low birthweight23, reduced gestational age24, preterm delivery25, and small-for-gestational age22,26,27. The evidence for low to moderate alcohol use in pregnancy has either been inconclusive28 or shown no increased risk for these adverse pregnancy outcomes29. Alcohol use in pregnancy has the most well established adverse fetal health effects30–32 and is associated with the development of fetal alcohol spectrum disorders33–35 and adverse neurodevelopmental outcomes36. In addition, prenatal drinking is associated with long-term effects, such as cognitive and behavioral challenges37,38, adverse speech and language outcomes39, executive functioning deficits in children40, and psychosocial consequences in adulthood41.\n\nSmoking during pregnancy exerts direct adverse effects on birth outcomes, including damage to the umbilical cord structure42, miscarriage43, increased risk for ectopic pregnancy44, low birthweight45–47, placental abruption45,46,48, preterm birth45,49, and increased infant mortality45,46,48. Also of concern are the deleterious health effects of second-hand smoke on newborns, which include higher rates of respiratory and ear infections, sudden infant death syndrome, behavioral dysfunction and cognitive impairment50. Additionally, women who were smokers before pregnancy might stop breastfeeding early so that they can take up smoking again51.\n\nSome pregnant women view cannabis use as harmless in pregnancy52; however, it has been linked with several deleterious effects, including preterm labor, low birthweight, small-for-gestational age, and admission to the neonatal intensive care unit53. Prenatal cannabis use has also been linked with adverse consequences for the growth of fetal and adolescent brains52, reduced attention and executive functioning skills, poorer academic achievement and more behavioral problems54. The adverse effects of marijuana are frequently observed with comorbid substance use, and are greatest in heavy users.\n\nThe extent of the adverse effects of cocaine use in pregnancy has been overestimated at times. However, there have been several large and thorough studies recently, which have all identified several risk factors associated with cocaine use during pregnancy, including premature rupture of membranes, placental abruption, preterm birth, low birthweight, and small for gestational age infants55,56. There have been inconsistent reports on the long-term effects of prenatal cocaine exposure on language, motor, and cognitive development, with a few studies describing positive findings57,58 and some studies reporting very little or no effects59. This inconsistency is probably connected to the confounding effects of the postnatal environment, including unsteady and disordered home environments, dysfunctional parenting, and heavy maternal polysubstance use60–62. Similar to cocaine use in pregnancy, methamphetamine use is linked with shorter gestational ages, lower birthweight63, fetal loss64, developmental and behavioral defects65, preeclampsia, gestational hypertension, and intrauterine fetal death66.\n\nOpioid use in pregnancy is correlated with a greater risk of low birthweight, respiratory problems, third trimester bleeding, toxemia and mortality12,67. Maternal opiate use is associated with an increased risk of neonatal abstinence syndrome (NAS), whereby opiate exposure in utero triggers a postnatal withdrawal syndrome12. Anywhere from 45 to 94% of infants exposed to opioids in utero, including methadone and buprenorphine, can be affected by NAS12. NAS results in substantial neonatal morbidity and increased healthcare utilization12,67, and consists of an array of signs and symptoms, including irritability, feeding difficulties, tremors, hypertonia, emesis, loose stools, seizures, and respiratory distress68. Opioid exposure in pregnancy has also been associated with postnatal growth deficiency, microcephaly, neurobehavioral problems, and sudden infant death syndrome67. Cigarette smoking, which is very common in pregnant women with an opioid use disorder (77–95%)69,70, may confound the effect of opioid use on poor pregnancy outcomes.\n\nA significant point to take into account is that the undesirable consequences of prenatal substance use are confounded by the frequency of coexisting substance use and comorbid psychiatric illness71,72. Women with substance use disorders also frequently experience inadequate prenatal care, poor nutrition, chronic medical problems, poverty, and domestic violence73,74. Furthermore, substance use in pregnancy may also result in an early dysfunctional maternal-infant relationship that can potentiate the negative effects of prenatal drug exposure60,61.\n\n\nTreatment of substance use in pregnancy\n\nThere are only a small number of effective therapies for substance use in pregnancy, which primarily involve behavioral counseling (see Table 1). Brief interventions75, in particular those that utilize motivational interviewing76,77, have been shown to reduce prenatal alcohol use. A recent randomized trial utilizing a telephone-based brief intervention suggests that this method may achieve similar results to the in-person intervention method of moderating prenatal drinking78. Some additional interventions to reduce prenatal drinking that have recently been described include screening via non-healthcare community workers79, counseling by midwives80, and multimedia and educational efforts aimed at improving awareness81.\n\nAs with alcohol, behavioral counseling is the main treatment for smoking cessation and relapse prevention in pregnant women. Unfortunately, psychotherapeutic interventions have had only moderate success82–85. Pharmacological treatments for smoking cessation have not been evaluated with respect to their safety and efficacy in pregnant and postpartum women82,86. Randomized clinical trials with nicotine replacement therapy in pregnant women have demonstrated limited efficacy in increasing the rates of abstinence87–90. The most successful intervention for prenatal smoking cessation is contingency management (CM) with financial incentives91–93, which has also reportedly improved birth outcomes94.\n\nTreatments specifically aimed at prenatal cannabis use are lacking. The current recommendation for lowering the use of cannabis in pregnancy includes the screening of pregnant women to increase the early identification of cannabis use52. Motivational interviewing (MI)95,96, cognitive-behavioral therapy (CBT)95–99, and CM therapies have had some success in reducing marijuana use in women, but they have not been evaluated specifically with pregnant users. Thus, novel interventions that explicitly target cannabis use are vital, particularly given the current tendency towards marijuana legalization.\n\nExisting evidence-based treatments for cocaine use in pregnancy include CBT, MI and CM100. As with smoking, CM is the intervention that shows most potential for treating cocaine-using pregnant women62. A randomized trial found that CM was associated with much longer duration of cocaine abstinence, higher number of cocaine-negative urine tests, and a greater proportion of documented abstinence when compared to community reinforcement approach and twelve-step facilitation101. Currently, there are no evidence-based pharmacological treatments for prenatal cocaine use. Nevertheless, a recent randomized, placebo-controlled trial supports the use of oral micronized progesterone as an intervention for postpartum cocaine use102. The study showed that women randomized to placebo had more self-reported cocaine use compared to women receiving micronized progesterone during the 12 weeks of the trial102. While these are preliminary findings and will require confirmation in a larger clinical trial, they show promise for the application of progesterone in postpartum women to reduce their cocaine use. Treatments for other stimulant use, such as methamphetamine, are limited. Research into reinforcement-based therapy (RBT) combined with a women-focused intervention among pregnant methamphetamine users reported a reduction in methamphetamine use over time103. However, there were no substantial distinctions between the intervention and control conditions103, not unlike another study using RBT to treat stimulant use in pregnancy104. RBT seems to have potential as an intervention for methamphetamine use but more research is required.\n\nMethadone maintenance is the standard care for pregnant women with opiate use disorders105. Conversion from illicit opioid use to opioid maintenance therapy in a medically supervised setting decreases maternal and neonatal morbidity. Methadone maintenance offers greater relapse prevention with a steady opioid dosing regimen, reduces risk-taking behavior, enhances compliance with prenatal care, and leads to better neonatal outcomes106. On the other hand, medication-assisted withdrawal, that is detoxification by gradually reducing the dose of an opioid substitute medication, is associated with a high opioid relapse rate and higher fetal morbidity and mortality rates106. Buprenorphine has recently emerged as another potential therapy for opioid use in pregnancy. A randomized controlled trial that compared methadone and buprenorphine in pregnant opioid users showed that infants whose mothers received buprenorphine needed less treatment for NAS, substantially lower doses of morphine to treat NAS symptoms, and had shorter stays in hospital, compared to the infants of women given methadone107. Notably, buprenorphine had lower retention rates with flexibly delivered doses and low fixed doses compared to methadone108. However, buprenorphine and methadone are equally effective when given as fixed medium or high doses108. CM has likewise been reported to be effective in treating opioid use in pregnancy, by significantly increasing abstinence and treatment attendance compared to controls109. Thus, CM appears to be an important addition to methadone or buprenorphine treatment in pregnant women.\n\n\nBreastfeeding and postpartum substance use\n\nBreastfeeding has the potential to be a useful tool for substance use in the postpartum period. Breastfeeding is the only available intervention shown to reduce NAS severity in opioid-exposed newborns110,111. Breastfeeding might also be protective for postpartum relapse. For example, among breastfeeding smokers, 10% stop breastfeeding because of smoking, and over half of recent or current smokers reported that smoking affected their infant feeding decision112. In addition, non-current smokers are more likely to initiate and continue breastfeeding compared to current smokers113,114. Therefore, the promotion of breastfeeding might prevent or delay postpartum relapse.\n\nWhile studies evaluating the potential role of breastfeeding as an intervention for substance use postpartum are limited, the rationale for such interventions is clear. Lactation reduces the HPA response to physical stress115. A behavior that promotes relaxation and reduces stress would be helpful to women with substance use disorders since psychosocial stress increases cravings116. While hormones released during lactation may mediate stress reduction, such hormones have other properties that may help women cope with addiction. Considerable attention has been dedicated to oxytocin, a hormone released during delivery and lactation. Oxytocin administration is under investigation for treatment of drug and alcohol use disorders116–118. In addition, lactation is positively associated with cognitive and motor development in the infant119. It is well known that stable attachment among children increases resiliency and protects against the development of addiction later in life120,121. Thus, an intervention that promotes lactation and intimacy through skin-to-skin contact may enhance stable attachment, and have the intergenerational benefit of protecting offspring from the development of addictive and other problematic behaviors120,122,123.\n\n\nConclusions\n\nSubstance use in pregnancy remains a significant public health problem, which can lead to several harmful maternal and neonatal outcomes. Which drug is being used and the degree of use, as well as the point of exposure, all influence the effects of drug use in pregnancy. In addition to the direct effects of drug exposure in utero, several other variables are associated with deleterious maternal and infant consequences, including psychiatric comorbidity, polysubstance use, limited prenatal care, environmental stressors and disrupted parental care. In conjunction, these factors can negatively influence pregnancy and infant outcomes, and should be taken in to account when developing interventions for prenatal substance use treatments. Many of the health problems associated with substance use in the prenatal period could be avoided given effective and well-timed medical care or intervention. Empirically-driven interventions for prenatal substance are needed. While there are few treatment options for substance use in pregnancy, CM seems to show the greatest promise as an effective therapy for the substances in which it has been studied. Future research needs to focus on developing tailored, safe, and acceptable treatments that can capitalize on pregnancy as a “teachable” moment that can motivate women to adopt risk-reducing health behaviors124–127.",
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PubMed Abstract | Publisher Full Text\n\nHeckman CJ, Egleston BL, Hofmann MT: Efficacy of motivational interviewing for smoking cessation: a systematic review and meta-analysis. Tob Control. 2010; 19(5): 410–416. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLevitt C, Shaw E, Wong S, et al.: Systematic review of the literature on postpartum care: effectiveness of interventions for smoking relapse prevention, cessation, and reduction in postpartum women. Birth. 2007; 34(4): 341–347. PubMed Abstract | Publisher Full Text\n\nReitzel LR, Vidrine JI, Businelle MS, et al.: Preventing postpartum smoking relapse among diverse low-income women: a randomized clinical trial. Nicotine Tob Res. 2010; 12(4): 326–335. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOncken CA, Kranzler HR: What do we know about the role of pharmacotherapy for smoking cessation before or during pregnancy? Nicotine Tob Res. 2009; 11(11): 1265–1273. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEssex HN, Parrott S, Wu Q, et al.: Cost-Effectiveness of Nicotine Patches for Smoking Cessation in Pregnancy: A Placebo Randomized Controlled Trial (SNAP). Nicotine Tob Res. 2015. 17(6): 636–42. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nEl-Mohandes AA, Windsor R, Tan S, et al.: A randomized clinical trial of trans-dermal nicotine replacement in pregnant African-American smokers. Matern Child Health J. 2013; 17(5): 897–906. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCooper S, Lewis S, Thornton JG, et al.: The SNAP trial: a randomised placebo-controlled trial of nicotine replacement therapy in pregnancy--clinical effectiveness and safety until 2 years after delivery, with economic evaluation. Health Technol Assess. 2014; 18(54): 1–128. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nColeman T, Cooper S, Thornton JG, et al.: A randomized trial of nicotine-replacement therapy patches in pregnancy. N Engl J Med. 2012; 366(9): 808–818. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nChamberlain C, O'Mara-Eves A, Oliver S, et al.: Psychosocial interventions for supporting women to stop smoking in pregnancy. Cochrane Database Syst Rev. 2013; 10: CD001055. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHiggins ST, Washio Y, Heil SH, et al.: Financial incentives for smoking cessation among pregnant and newly postpartum women. Prev Med. 2012; 55(Suppl): S33–S40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIerfino D, Mantzari E, Hirst J, et al.: Financial incentives for smoking cessation in pregnancy: a single-arm intervention study assessing cessation and gaming. Addiction. 2015; 110(4): 680–688. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHiggins ST, Bernstein IM, Washio Y, et al.: Effects of smoking cessation with voucher-based contingency management on birth outcomes. Addiction. 2010; 105(11): 2023–2030. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHoch E, Bühringer G, Pixa A, et al.: CANDIS treatment program for cannabis use disorders: findings from a randomized multi-site translational trial. Drug Alcohol Depend. 2014; 134: 185–193. PubMed Abstract | Publisher Full Text\n\nHoch E, Noack R, Henker J, et al.: Efficacy of a targeted cognitive-behavioral treatment program for cannabis use disorders (CANDIS). Eur Neuropsychopharmacol. 2012; 22(4): 267–280. PubMed Abstract | Publisher Full Text\n\nStephens RS, Roffman RA, Simpson EE: Treating adult marijuana dependence: a test of the relapse prevention model. J Consult Clin Psychol. 1994; 62(1): 92–99. PubMed Abstract | Publisher Full Text\n\nCarroll KM, Nich C, Lapaglia DM, et al.: Combining cognitive behavioral therapy and contingency management to enhance their effects in treating cannabis dependence: less can be more, more or less. Addiction. 2012; 17(9): 1650–1659. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCopeland J, Swift W, Roffman R, et al.: A randomized controlled trial of brief cognitive-behavioral interventions for cannabis use disorder. J Subst Abuse Treat. 2001; 21(2): 55–64; discussion 65–6. PubMed Abstract | Publisher Full Text\n\nTerplan M, Ramanadhan S, Locke A, et al.: Psychosocial interventions for pregnant women in outpatient illicit drug treatment programs compared to other interventions. Cochrane Database Syst Rev. 2015; 4: Cd006037. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSchottenfeld RS, Moore B, Pantalon MV: Contingency management with community reinforcement approach or twelve-step facilitation drug counseling for cocaine dependent pregnant women or women with young children. Drug Alcohol Depend. 2011; 118(1): 48–55. PubMed Abstract | Publisher Full Text\n\nYonkers KA, Forray A, Nich C, et al.: Progesterone Reduces Cocaine Use in Postpartum Women with a Cocaine Use Disorder: A Randomized, Double-Blind Study. Lancet Psychiatry. 2014; 1(5): 360–367. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones HE, Myers B, O'Grady KE, et al.: Initial feasibility and acceptability of a comprehensive intervention for methamphetamine-using pregnant women in South Africa. Psychiatry J. 2014; 2014: 929767. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nMattick RP, Breen C, Kimber J, et al.: Buprenorphine maintenance versus placebo or methadone maintenance for opioid dependence. Cochrane Database Syst Rev. 2014; 2: CD002207. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nJones HE, Haug N, Silverman K, et al.: The effectiveness of incentives in enhancing treatment attendance and drug abstinence in methadone-maintained pregnant women. Drug Alcohol Depend. 2001; 61(3): 297–306. PubMed Abstract | Publisher Full Text\n\nO'Connor AB, Collett A, Alto WA, et al.: Breastfeeding rates and the relationship between breastfeeding and neonatal abstinence syndrome in women maintained on buprenorphine during pregnancy. J Midwifery Womens Health. 2013; 58(4): 383–388. PubMed Abstract | Publisher Full Text\n\nWelle-Strand GK, Skurtveit S, Jansson LM, et al.: Breastfeeding reduces the need for withdrawal treatment in opioid-exposed infants. 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PubMed Abstract | Publisher Full Text\n\nMcRae-Clark AL, Carter RE, Price KL, et al.: Stress- and cue-elicited craving and reactivity in marijuana-dependent individuals. Psychopharmacology (Berl). 2011; 218(1): 49–58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPedersen CA, Smedley KL, Leserman J, et al.: Intranasal oxytocin blocks alcohol withdrawal in human subjects. Alcohol Clin Exp Res. 2013; 37(3): 484–489. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee MR, Glassman M, King-Casas B, et al.: Complexity of oxytocin's effects in a chronic cocaine dependent population. Eur Neuropsychopharmacol. 2014; 24(9): 1483–1491. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBernard JY, De Agostini M, Forhan A, et al.: Breastfeeding duration and cognitive development at 2 and 3 years of age in the EDEN mother-child cohort. J Pediatr. 2013; 163(1): 36–42.e1. 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PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nColman GJ, Joyce T: Trends in smoking before, during, and after pregnancy in ten states. Am J Prev Med. 2003; 24(1): 29–35. PubMed Abstract | Publisher Full Text\n\nHeil SH, Herrmann ES, Badger GJ, et al.: Examining the timing of changes in cigarette smoking upon learning of pregnancy. Prev Med. 2014; 68: 58–61. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKitsantas P, Gaffney KF, Wu H, et al.: Determinants of alcohol cessation, reduction and no reduction during pregnancy. Arch Gynecol Obstet. 2014; 289(4): 771–779. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "13839",
"date": "13 May 2016",
"name": "Oscar Garcia-Algar",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13840",
"date": "13 May 2016",
"name": "Kirsten A. Donald",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-887
|
https://f1000research.com/articles/5-881/v1
|
12 May 16
|
{
"type": "Research Note",
"title": "Ultraviolet B, melanin and mitochondrial DNA: Photo-damage in human epidermal keratinocytes and melanocytes modulated by alpha-melanocyte-stimulating hormone",
"authors": [
"Markus Böhm",
"Helene Z. Hill",
"Markus Böhm"
],
"abstract": "Alpha-melanocyte-stimulating hormone (alpha-MSH) increases melanogenesis and protects from UV-induced DNA damage. However, its effect on mitochondrial DNA (mtDNA) damage is unknown. We have addressed this issue in a pilot study using human epidermal keratinocytes and melanocytes incubated with alpha-MSH and irradiated with UVB. Real-time touchdown PCR was used to quantify total and deleted mtDNA. The deletion detected encompassed the common deletion but was more sensitive to detection. There were 4.4 times more mtDNA copies in keratinocytes than in melanocytes. Irradiation alone did not affect copy numbers. Alpha-MSH slightly increased copy numbers in both cell types in the absence of UVB and caused a similar small decrease in copy number with dose in both cell types. Deleted copies were nearly twice as frequent in keratinocytes as in melanocytes. Alpha-MSH reduced the frequency of deleted copies by half in keratinocytes but not in melanocytes. UVB dose dependently led to an increase in the deleted copy number in alpha-MSH-treated melanocytes. UVB irradiation had little effect on deleted copy number in alpha-MSH-treated keratinocytes. In summary, alpha-MSH enhances mtDNA damage in melanocytes presumably by increased melanogenesis, while α-MSH is protective in keratinocytes, the more so in the absence of irradiation.",
"keywords": [
"Melanin",
"alpha-melanocyte stimulating hormone",
"UVB",
") melanocytes",
"keratinocytes",
"mitochondrial DNA",
"mitochondrial DNA deletions",
"polymerase chain reaction"
],
"content": "Introduction\n\nMelanin is found in the cell cytoplasm as are the mitochondria. Although the principal role of melanin is photoprotection, the pigment is well known to emit melanin free radicals and act as a photosensitizing agent1,2. These studies were undertaken to determine the effect of ultraviolet B irradiation (UVB) on the induction of reactive oxygen species damage of mitochondrial DNA (mtDNA) in cultures of pigment-induced human epidermal melanocytes compared to human epidermal keratinocytes. Deletions in mitochondrial DNA are the hallmark of reactive oxygen species damage.\n\n\nMaterials and methods\n\nNormal human neonatal epidermal melanocytes were purchased from Tebu-bio (Portland, OR) (catalog # 104-05n 5E5 Cells) and normal human neonatal epidermal keratinocytes were obtained from PromoCell (Heidelberg, Germany) (catalog # C-12007). Melanocytes and keratinocytes were routinely cultured as reported previously3. Melanocytes were maintained in MGM-M2 medium plus all supplements (Cascade Biologics, Portland, OR; M-254-500 plus S-002-5) while keratinocytes were cultured in KBM-2 medium with all supplements (PromoCell; C-20111). 500,000–700,000 cells were seeded into 6 cm diameter culture dishes. On the following day cells were deprived for 48 hrs from bovine pituitary extract followed by pre-incubation with 10-6 M α-MSH (α-melanocyte-stimulating hormone) (Calbiochem, Schwalbach, Germany) for 6 hrs at 37°C.\n\nUVB treatment was performed with an irradiation bank consisting of six fluorescent bulbs (TL12, Philips) which emit most of their energy within the UVB range with an emission peak of 313 nm. Cells were irradiated through phosphate-buffered saline at 5, 10 and 15 mJ/cm2 followed by incubation with experimental medium (5 ml) with and without α-MSH (10-6 M) for 24 hrs.\n\nDNA was extracted using the Epicentre kit from Biozym (Hess. Oldendorf, Germany; MCD85201). DNA was finally dissolved in 10 mM Tris buffer with 0.1 mM EDTA.\n\nFive micrograms of each purified DNA sample were incubated with 10 units of AflIII (New England BioLabs, Ipswich, MA) for 1 hour at 37°C according to the manufacturer’s directions. The reaction mixture was repurified on spin columns according to the manufacturer’s directions (DNA Clean and Concentrator columns, Zymo Research Corporation, Orange, CA) and diluted appropriately in 10 mM Tris pH 8.5.\n\nThe Roche LightCycler-2 (Roche Applied Science, Indianapolis, IN) was used throughout these studies. Total mitochondrial genomes were quantified using primers HSSN1307 and HSAS 14334 (5’GTACCCACGTAAAGACGTTAGG3’ and 5’TACTGCTAAATCCACC-TTCG3’ respectively) and probe 5’FAM-CCCATGAGGCAAGAAATT-BHQ1-3’3. Each capillary contained 250 pg DNA, 300 nM each primer, 200 nM probe, 0.01 μL uracil-DNAglycosylase, heat-labile (Roche) (UNG) and 1X LightCycler TaqMan Master Mix in a total volume of 5 μL. The Master Mix contains dUTP in place of dTTP in order for the amplified product to contain U in place of T. Pre-incubation of the PCR mixture with UNG allows for the degradation of any carryover contaminants from earlier reactions. The cycling protocol called for 10 min at 35°C for UNG digestion, followed by 10 min at 95°C to inactivate UNG and activate the Taq polymerase; up to 40 cycles of 95°C 10 sec for denaturation, 60°C 20 sec for annealing, 72°C 10 sec for polymerization and acquisition. Each run also contained two capillaries of 1 × 105 copies of the plasmid pKW into which the Total amplicon had been inserted4.\n\nPrimers for the deletion were HSSN8416 and HSAS8542 (5’-CCTTACACTATTCCT-CATCACC-3’ and 5’-TGTGGTCTTTGGAGTAGAAACC-3’ respectively)3. The probe was 5’-6FAM-TGGCAGCCTAGCATTAGCAGTT-BHQ1-3’)4. Each capillary contained 10 ng sample DNA, primers, probe, UNG and Master Mix as in the Total reactions. Glycosylation and denaturation were as for the Total PCR. Amplification was initiated with 5 cycles of Touchdown PCR: cycle 1: 95°C for 10 sec, 65°C for 5 sec, dropping 1 degree per cycle down to 60°C followed by no more than 55 cycles of 95°C for 10 sec, 60°C for 15 sec. Results with touchdown PCR were more reproducible and took fewer cycles to reach the crossing point5. The touchdown amplicon was sequenced in the Molecular Resource Facility at the NJ Medical School.\n\nThe results shown are averages of three separate experiments. The proprietary software accompanying the Roche LightCycler was used to determine the crossing point of each sample. The crossing points of the samples were compared to crossing points of known copy numbers of standards in order to calculate sample copy numbers. In each experiment, total copies were determined in triplicate and deleted copies were determined in quintuplicate. Please see Dataset 1.\n\n\nResults\n\nGel analysis determined that the touchdown amplicon was smaller than that of the common deletion (CD): less than 100 bp versus 127 bp. The CD is 4977 bp and spans bp 8470 to bp 13447. The touchdown deletion (TD) spans 5021 bp from bp 8433 to bp 13454. The CD is contained within the TD. Just as the CD is based on repeat sequences, in this case, of 13 bp, ACCTCCCTCACCA, the TD is based on the 5 bp repeat TCACC. Note that the 5 bp repeat is actually contained in the 13 bp repeat and the 3’ ends of both deletions coincide. The 5’ ends of the two deletions are 37 bp apart.\n\nFigure 1 shows the effect of UVB irradiation on total mitochondrial genome copy numbers in melanocytes and keratinocytes that have been treated with α-MSH, compared to untreated (no α-MSH) controls. In the absence of any α-MSH, keratinocytes have 4.4 times more mtDNA copies than melanocytes. Copy numbers of mtDNA from non-α-MSH treated cells of both cell types are unaffected by UVB exposure in this dose range. α-MSH causes a small increase in copy number in both cell types in the absence of irradiation and a parallel decline with dose.\n\nBlack symbols: melanocytes, red symbols: keratinocytes. Circles: no α-MSH; squares: + α-MSH.\n\nFigure 2 shows the effect of UVB irradiation on deleted copy numbers in melanocytes and keratinocytes that have been exposed to α-MSH compared to non-α-MSH treated controls. In the absence of any α-MSH treatment, deleted copies are about half as frequent in melanocytes than in keratinocytes. In the absence of α-MSH treatment, deleted copies decline slightly with increasing radiation dose in both types of cell. This is more likely due to slower replication of the deleted genomes than to their direct loss.\n\nBlack symbols: melanocytes, red symbols: keratinocytes. Circles: no α-MSH; squares: + α-MSH.\n\nIn melanocytes pre-treated with α-MSH, there is a marked increase in the frequency of deleted genomes with increasing UVB dose. This is likely to be due to the production of reactive oxygen species from UVB interaction with the melanin pigment or with melanin precursors. α-MSH actually causes a decrease in deleted copies in unexposed keratinocytes suggesting that, after exposure deleted copies replicate more slowly or that some protective agent has been induced. This effect is lessened as the UVB dose increases.\n\n\nDiscussion\n\nThe pigment melanin is widely distributed in the animal world and has multiple functions. In the skin, it is both photoprotective6–8 and photosensitizing9–13. The quixotic nature of melanin has been the subject of a number of studies and reviews2,14–18.\n\nα-MSH is a key melanotropic factor induced by UVB irradiation in keratinocytes to turn on melanogenesis. Of note, we and others have shown that α-MSH is capable of reducing UVB-mediated DNA damage in both human melanocytes and keratinocytes independent from its melanin-inducing effect19–21. Moreover, we recently found that α-MSH has indirect anti-oxidative effects on human melanocytes and keratinocytes since it upregulates expression of the transcription factor Nrf2, a master regulator of phase II detoxifying enzymes3. Thus, we speculated that in both cell types α-MSH may also protect from some UVB-induced oxidative effects on mtDNA. The CD in mtDNA is a hallmark for oxidative stress. Arck et al. reported more CDs in graying hair follicles than in unpigmented follicles22, suggesting that the pigment was responsible for the effect. Our findings in fact suggest that α-MSH-induced melanin synthesis acts as a photosensitizer for mtDNA damage in the form of the TD after irradiation with UVB in the MED (minimal erythema dose) range. The presence of melanin and/or its precursors in the cytoplasm may indeed produce oxidative damage in mtDNA which is enhanced by solar irradiation, thus altering the “milieu intérieur” and possibly leading to altered mitochondrial function. In this context, it should be very interesting to determine the direct influence of α-MSH alone or in combination with UVB on mitochondrial metabolism and biogenesis. In addition, investigation on the impact of signaling-deficient MC1R alleles on mtDNA damage in melanocytes treated with α-MSH and UVB may disclose unexpected clues in the pathogenesis of cutaneous melanoma.\n\nRecently, Premi et al.23 irradiated mouse and human pigmented and non-pigmented cells with UVA and measured the production of cyclobutane pyrimidine dimers (CPDs) in total cellular DNA. They found that CPDs continued to increase in the pigmented cells, but not in the non-pigmented cells, for as long as 3 hours after irradiation, after which they declined due to repair. In our study, deletions were measured at only a single time point so there is no way of determining the dynamics of formation. However, once formed, they would persist unrepaired. It would be of interest to study the time-dependent effects of UVB and UVA on induction of mitochondrial deletions in unpigmented, pigmenting and pigmented cells, more especially since Boulton and Birch-Machin recently found that pigment is protective against reactive oxygen generation by complex II24. Another demonstration, perhaps, of the dual nature of melanin as a two-edged sword15. Function is protected while genetic information is damaged.\n\n\nConclusion\n\nThere are about 2.4 times more deleted copies relative to total copies in melanocytes compared to keratinocytes. UVB increases the ratio in the presence of α-MSH in melanocytes while it remains relatively stable in keratinocytes. This suggests that α-MSH causes a destabilization of the free-radical balance in melanocytes but not in keratinocytes. This effect may be the result of the increase in melanin precursors and/or pigment by the presence of α-MSH.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for ‘Ultraviolet B, melanin and mitochondrial DNA: Photo-damage in human epidermal keratinocytes and melanocytes modulated by alpha-melanocyte-stimulating hormone’, Böhm and Hill 2016, 10.5256/f1000research.8582.d12145425",
"appendix": "Author contributions\n\n\n\nMB and HZH together conceived the study. MB directed the cell culture, irradiations, cell harvesting and DNA extractions. HZH restricted, repurified the DNA and performed the Real Time PCR. HZH and MB contributed equally to the manuscript preparation and agree 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 are grateful for expert technical assistance by Mara Apel.\n\n\nReferences\n\nCollins B, Poehler TO, Bryden WA: EPR persistence measurements of UV-induced melanin free radicals in whole skin. Photochem Photobiol. 1995; 62(3): 557–560. PubMed Abstract | Publisher Full Text\n\nMeredith P, Sarna T: The physical and chemical properties of eumelanin. Pigment Cell Res. 2006; 19(6): 572–594. 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PubMed Abstract | Publisher Full Text\n\nMarrot L, Belaidi JP, Meunier JR, et al.: The human melanocyte as a particular target for UVA radiation and an endpoint for photoprotection assessment. Photochem Photobiol. 1999; 69(6): 686–693. PubMed Abstract | Publisher Full Text\n\nTakeuchi S, Zhang W, Wakamatsu K, et al.: Melanin acts as a potent UVB photosensitizer to cause an atypical mode of cell death in murine skin. Proc Natl Acad Sci USA. 2004; 101(42): 15076–15081. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang HT, Choi B, Tang MS: Melanocytes are deficient in repair of oxidative DNA damage and UV-induced photoproducts. Proc Natl Acad Sci USA. 2010; 107(27): 12180–12185. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKipp C, Young AR: The soluble eumelanin precursor 5,6-dihydroxyindole-2-carboxylic acid enhances oxidative damage in human keratinocyte DNA after UVA irradiation. Photochem Photobiol. 1999; 70(2): 191–198. PubMed Abstract | Publisher Full Text\n\nHill HZ: The function of melanin or six blind people examine an elephant. Bioessays. 1992; 14(1): 49–56. PubMed Abstract | Publisher Full Text\n\nHill HZ, Li W, Xin P, et al.: Melanin: a two edged sword? Pigment Cell Res. 1997; 10(3): 158–161. PubMed Abstract | Publisher Full Text\n\nHill HZ, Hill GJ, Cieszka K, et al.: Comparative action spectrum for ultraviolet light killing of mouse melanocytes from different genetic coat color backgrounds. Photochem Photobiol. 1997; 65(6): 983–989. PubMed Abstract | Publisher Full Text\n\nHill HZ, Hill GJ: UVA, pheomelanin and the carcinogenesis of melanoma. Pigment Cell Res. 2000; 13(Suppl 8): 140–144. PubMed Abstract | Publisher Full Text\n\nGoodman G, Bercovich D: Melanin directly converts light for vertebrate metabolic use: heuristic thoughts on birds, Icarus and dark human skin. Med Hypotheses. 2008; 71(2): 190–202. PubMed Abstract | Publisher Full Text\n\nBöhm M, Wolff I, Scholzen TE, et al.: alpha-Melanocyte-stimulating hormone protects from ultraviolet radiation-induced apoptosis and DNA damage. J Biol Chem. 2005; 280(7): 5795–5802. PubMed Abstract | Publisher Full Text\n\nKadekaro AL, Kavanagh R, Kanto H, et al.: alpha-Melanocortin and endothelin-1 activate antiapoptotic pathways and reduce DNA damage in human melanocytes. Cancer Res. 2005; 65(10): 4292–4299. PubMed Abstract | Publisher Full Text\n\nDong L, Wen J, Pier E, et al.: Melanocyte-stimulating hormone directly enhances UV-induced DNA repair in keratinocytes by a xeroderma pigmentosum group A-dependent mechanism. Cancer Res. 2010; 70(9): 3547–3556. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArck PC, Overall R, Spatz K, et al.: Towards a \"free radical theory of graying\": melanocyte apoptosis in the aging human hair follicle is an indicator of oxidative stress induced tissue damage. FASEB J. 2006; 20(9): 1567–1569. PubMed Abstract | Publisher Full Text\n\nPremi S, Wallisch S, Mano CM, et al.: Photochemistry. Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure. Science. 2015; 347(6224): 842–847. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoulton SJ, Birch-Machin MA: Impact of hyperpigmentation on superoxide flux and melanoma cell metabolism at mitochondrial complex II. FASEB J. 2015; 29(1): 346–53. PubMed Abstract | Publisher Full Text\n\nBöhm M, Hill HZ: Dataset 1 in: Ultraviolet B, melanin and mitochondrial DNA: Photo-damage in human epidermal keratinocytes and melanocytes modulated by alpha-melanocyte-stimulating hormone. F1000Research. 2016. Data Source"
}
|
[
{
"id": "13934",
"date": "23 May 2016",
"name": "Andrzej Slominski",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is well performed study by experts in the field. The methodology and data collection are sound and data properly discussed.\nI believe that that careful readers would like to have more extended list of references plus appropriate comments in the introduction and discussion.\nFor example on the capability of the skin to express POMC and its processing to MSH, ACTH and endorphin peptides1, on hormonal and nutritional regulation of melanin pigmentation with regulatory consequences,2,3 and double-edge sword role of melanin in melanoma4.",
"responses": []
},
{
"id": "14092",
"date": "01 Jun 2016",
"name": "Jose Carlos Garcia-Borron",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report from Markus Böhm and Helene Hill describes a study on the mitochondrial DNA damaging effect of ultraviolet B (UVB) radiation in keratinocytes and melanocytes, in the presence or absence of the melanotropic peptide hormone alphaMSH. It is known that binding of this melanocortin to its cognate receptor expressed in melanocytes, the MC1R, triggers a series of pleiotropic responses notably including activation of the melanogenic pathway and induction of antioxidant enzymes as well as DNA repair pathways. it is also known that MC1R is expressed in many nonmelanocytic cell types including keratinocytes. In fact, work in M Böhm’s lab has been crucial to the current view of alphaMSH/MC1R signaling as a key photoprotective process, and has led the field of extramelanocytic actions of MC1R. The contributions of HZ Hill have been also important to appreciate the dual character of melanins, with photoprotective roles but also endowed with a photosensitizing potential. Accordingly, the work is presented by leading experts in the field.\nWhereas the role of alphaMSH in mediating protection of nuclear DNA against UVB is well established, not much is known about a possible action on mitochondrial DNA. Therefore, the paper has the merit to address a still unexplored question. The authors report a higher frequency of damaged mitochondrial DNA in control keratinocytes not exposed to UVB, compared with control melanocytes. Rather counterintuitively, alphaMSH apparently protects mitochondrial DNA against UVB-induced damage in keratinocytes, whereas it promotes an increase in the number of damaged copies in melanocytes. Since the hormone stimulates melanogenesis in melanocytes but not in keratinocytes, this suggests that the melanin polymer or some unidentified melanogenic intermediates might in fact act as photosensitizers, in keeping with the two-edged sword character of melanin emphasized by previous contributions from HZ Hill.\nThe experiments reported here are well described and most likely well conducted, and I have not detected any overinterpretation of the results. One caveat that leaves room for improvement is the absence of a statistical analysis of the small differences between the different experimental conditions.\nA major strength of this short report is that it raises new questions that are likely to be addressed in the near future. Do eumelanin-containing melanocytes from dark-skinned donors display the same responses to UVB in terms of mitochondrial DNA damage as feomelanogenic melanocytes from individuals with red hair color? Do melanocytes expressing the consensus MC1R respond to alphaMSH comparably to melanocytes expressing variant MC1R? Is there a differential effect of alphaMSH in melanocytes challenged with UVB in the presence of inhibitors of the rate-limiting melanogenic enzyme tyrosinase, or for that matter in albino melanocytes? And what is the impact of UVB and alphaMSH signaling on the performance of oxidative phosphorylation/electron transport chain as a main source of DNA-damaging reactive oxygen species? Some of these questions are mentioned in the short but well focused discussion, and their answer will contribute to a better understanding of the complex role of pigment production in the homeostasis of UVB-irradiated human skin.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-881
|
https://f1000research.com/articles/5-880/v1
|
12 May 16
|
{
"type": "Review",
"title": "Central control of body temperature",
"authors": [
"Shaun F. Morrison"
],
"abstract": "Central neural circuits orchestrate the behavioral and autonomic repertoire that maintains body temperature during environmental temperature challenges and alters body temperature during the inflammatory response and behavioral states and in response to declining energy homeostasis. This review summarizes the central nervous system circuit mechanisms controlling the principal thermoeffectors for body temperature regulation: cutaneous vasoconstriction regulating heat loss and shivering and brown adipose tissue for thermogenesis. The activation of these thermoeffectors is regulated by parallel but distinct efferent pathways within the central nervous system that share a common peripheral thermal sensory input. The model for the neural circuit mechanism underlying central thermoregulatory control provides a useful platform for further understanding of the functional organization of central thermoregulation, for elucidating the hypothalamic circuitry and neurotransmitters involved in body temperature regulation, and for the discovery of novel therapeutic approaches to modulating body temperature and energy homeostasis.",
"keywords": [
"Brown adipose tissue",
"shiver",
"cutaneous vasoconstriction",
"thermogenesis",
"fever",
"sympathetic nerve activity",
"preoptic hypothalamus",
"rostral raphe pallidus",
"dorsomedial hypothalamus",
"therapeutic hypothermia",
"obesity"
],
"content": "\n\nBody temperature (Tcore) is a critical homeostatic parameter influencing cellular function and organismal survival. Life-threatening protein denaturation looms as Tcore increases, and reductions in membrane fluidity, ion fluxes, and enzyme performance accompany significant reductions in Tcore. The fundamental central neural circuits for thermoregulation orchestrate behavioral and autonomic repertoires that maintain Tcore during thermal challenges, sensed by thermal receptors, that arise from either the ambient or the internal (e.g., during exercise) environment. A variety of other neural circuits and neurochemical modulators impinge on the fundamental thermoregulatory pathways to produce the alterations in Tcore that occur with circadian and ultradian periodicities1,2 and those that accompany many behavioral states, such as fever during sickness, increased Tcore during psychological stress and at ovulation, and reductions in Tcore during sleep, sepsis, or exposure to metabolic distress (e.g., hypoxia and starvation). Recent research in the central nervous system (CNS) control of Tcore focuses principally on: (a) elaborating the CNS pathways and neurotransmitter systems involved in the fundamental central thermoregulatory network; (b) the modulation of activity within the fundamental central thermoregulatory network by non-thermal factors and behavioral states; and (c) pharmacological manipulation of the CNS thermoregulatory network for a variety of therapeutic goals (e.g., reducing Tcore to produce therapeutic hypothermia in stroke victims).\n\nIn its simplest form, the fundamental thermoregulatory network can be modeled as a reflex3,4 in which a central integrative circuit alters the activity of thermoeffector mechanisms in response to an input from the combination of peripheral (i.e., skin) and central (i.e., visceral and brain) thermoreceptors that provides a consolidated assessment of Tcore and, importantly, of imminent threats to Tcore. The primary thermoeffector mechanisms recruited for both cold defense and centrally driven hyperthermias (e.g., fever) include: (a) thermoregulatory behaviors to reduce the loss of heat produced during basal metabolism; (b) cutaneous vasoconstriction (CVC) to conserve heat in the body core and limit heat loss to the environment; and (c) heat production (thermogenesis). The principal sources of metabolic heat production, beyond those contributing to basal metabolic rate (e.g., pumping ions across membranes), are brown adipose tissue (BAT), whose sympathetic neural input fuels mitochondria that shunt proton fluxes into heat production, and shivering behavior in skeletal muscle, dependent on the inefficiency of ATP utilization to generate heat. Effector mechanisms for heat defense include: (a) thermoregulatory behavior to increase heat loss; (b) cutaneous vasodilation, which in humans includes a sympathetic vasodilator outflow5,6, combined with increased cardiac output7 and visceral vasoconstriction8–10 to facilitate core heat loss from the body surface; and (c) evaporative cooling (e.g., sweating).\n\nApart from important “first responders”, the CNS pathways controlling and mediating thermoregulatory behaviors remain incompletely defined11–15. Along these same lines, most research subjects are furry mammals that are strongly dependent on non-sweating mechanisms for evaporative cooling. Although this has limited the available detail on the CNS pathways regulating human sweating6,16,17, considerable insight has been gained on the CNS regulation of other mechanisms of evaporative cooling, such as panting18–20. Additionally, since most of the basic neuroscience of CNS thermoregulatory pathways has been derived from experiments in rodents, including the exclusively in vitro studies of neurons with intrinsic thermosensitivity, the translation of the conclusions, including the circuit model in Figure 1, to humans must be cautiously undertaken (e.g., 21). Finally, although considerable progress has been achieved in the relatively young field of the neuroscience of thermoregulation, the synthesis (Figure 1) of our understanding of this multifaceted neural network controlling multiple thermoeffectors represents a working model, with the expectation of revisions and added detail.\n\nCool and warm cutaneous thermoreceptors transmit signals to respective primary sensory neurons in the dorsal root ganglia (DRG) which relay this information to second-order thermal sensory neurons in the dorsal horn (DH). Cool sensory DH neurons glutamatergically activate third-order sensory neurons in the external lateral subnucleus of the lateral parabrachial nucleus (LPB), while warm sensory DH neurons project to third-order sensory neurons in the dorsal subnucleus of the LPB. Thermosensory signals driving thermoregulatory responses are transmitted from the LPB to the preoptic area (POA), which contains the microcircuitry through which cutaneous and core thermal signals are integrated to regulate the balance of POA outputs that are excitatory (dashed green) and inhibitory (dashed red) to thermogenesis-promoting neurons in the dorsomedial hypothalamus (DMH) and to CVC sympathetic premotor neurons in the rostral raphe pallidus (rRPa). Within the POA, GABAergic interneurons (red) in the median preoptic (MnPO) subnucleus are postulated to receive a glutamatergic input from skin cooling-activated neurons in LPB and inhibit each of the distinct populations of warm-sensitive (W-S) neurons in the medial preoptic area (MPA) that control CVC, BAT, and shivering. In contrast, glutamatergic interneurons (dark green) in the MnPO are postulated to be excited by glutamatergic inputs from skin warming-activated neurons in LPB and, in turn, excite the populations of W-S neurons in MPA. Prostaglandin E2 (PGE2) binds to EP3 receptors, which are postulated to inhibit the activity of each of the classes of W-S neurons in the POA. Preoptic W-S neurons may provide inhibitory control of CVC by inhibiting CVC sympathetic premotor neurons in the rostral ventromedial medulla, including the rRPa, that project to CVC sympathetic preganglionic neurons (SPNs) in the intermediolateral nucleus (IML). Preoptic W-S neurons may provide inhibitory thermoregulatory control of BAT and shivering thermogenesis by inhibiting BAT sympathoexcitatory neurons and shivering-promoting neurons, respectively, in the DMH, which, when disinhibited during skin and core cooling, provide respective excitatory drives to BAT sympathetic premotor neurons and to skeletal muscle shivering premotor neurons in the rRPa. These, in turn, project, respectively, to BAT SPNs in the IML and to alpha (α) and gamma (γ) motoneurons in the ventral horn (VH) of the spinal cord.\n\nThe CNS thermoregulatory control of the sympathetic outflows mediating CVC and BAT thermogenesis and of the somatic motoneurons producing shivering is effected through parallel but distinct, effector-specific, integrative/efferent circuits (Figure 1, and reviewed in 22–25) that share common peripheral thermal sensory inputs. The hypothalamus contains the primary integrative and rostral efferent components of these circuits. Although many details of the preoptic area (POA) microcircuitry for thermoregulation remain to be elucidated, neurons in the POA are postulated to integrate ascending peripheral thermosensory signals with local thermosensitivity to regulate the output of BAT and shivering thermogenesis-promoting neurons in the dorsomedial hypothalamus (DMH)26,27 and of CVC-promoting neurons in the median preoptic nucleus (MnPO)28,29.\n\nThe POA regulation of DMH thermogenesis-promoting neurons represents the balance between a GABAergic inhibition30,31 and a glutamatergic excitation32; the latter inputs, potentially arising from neurons in the MnPO that project to the DMH, are synaptically connected to BAT33 and express the leptin receptor34. These glutamatergic inputs to DMH32 could provide the excitation required to drive the BAT sympathoexcitatory neurons and the shivering-promoting neurons in DMH when their POA inhibitory input is reduced during skin cooling or fever35. Although intrinsically warm-sensitive (W-S) (Figure 1), POA neurons36–38, generally located in the medial preoptic area (MPA)39, are postulated to play a key role in central thermoregulation by providing a prominent core temperature-modulated, GABAergic38 regulation of thermogenesis-promoting neurons in DMH (Figure 1), the considerable direct functional evidence required to establish this attractive hypothesis has yet to be obtained. Different thermal sensitivities or neurochemical modulation among populations of temperature-sensitive POA neurons may underlie the differential responsiveness of different effectors to changes in cutaneous versus brain temperatures40 as well as the significant alterations in thermoeffector activation during different sleep phases41. Through their responses to immune signaling molecules, neurons in the POA are also the primary site for the organization and maintenance of the febrile response to inflammation and infection, which includes the stimulation of CVC, and shivering (“chills”) and BAT thermogenesis mediated by the action of prostaglandin E2 (PGE2) on its EP3 receptors42–45. Similarly, the fundamental thermoregulatory network mediates stress-induced hyperthermia46–48. Unraveling the complexity of the thermoregulatory circuitry in the hypothalamus20,28,29,49–52, including the phenotypic characterization of the projection neurons34 and their synaptic interactions that mediate the circadian13 and many behavioral53,54 modulations in Tcore, continues to pose significant research challenges for understanding the “heart” of the CNS thermoregulatory network. The downstream targets of the hypothalamic projection neurons for thermoregulation are the sympathetic and somatic premotor neurons in the rostral ventromedial medulla, centered on the rostral raphe pallidus (rRPa). Midbrain (e.g., periaqueductal gray55–57 and retrorubral field58) and medullary (e.g., ventrolateral medulla and nucleus of the solitary tract (NTS)59,60) pathways to these premotor neurons exert important modulatory influences on the thermoregulatory activation of thermoeffector organs (reviewed in 22,24). These premotor neurons, in turn, excite CVC and BAT sympathetic preganglionic neurons and α-motoneurons and γ-motoneurons61,62 in the spinal cord.\n\nCold and warm thermoreceptors in the skin and viscera provide the extracranial thermal signals relating to skin temperature and Tcore. These are integrated with the brain temperature information potentially derived from the discharge of W-S, GABAergic preoptic neurons of the central thermoregulatory network to regulate thermoeffector activities. The membranes of thermoreceptor afferent neurons contain transient receptor potential (TRP) cation channels whose temperature-dependent conductances transduce skin temperature into primary thermoreceptor afferent neuronal activity. The TRPM8 channel, activated by menthol and cooling, is the strongest candidate for the cutaneous cold receptor (reviewed in 63). The identity of the peripheral warm receptor remains to be established, but the warm-sensing mechanism of preoptic neurons, though still debated64–66, is unlikely to involve a transient receptor potential (TRP) channel67. Primary thermoreceptor dorsal root ganglion neurons synapse on thermoreceptive-specific, lamina I spinal (or trigeminal) dorsal horn cells68, and these, in turn, collateralize to innervate the thalamus, providing the neural substrate for cutaneous thermosensory perception and localization68,69, and the pontine lateral parabrachial nucleus (LPB)70,71, which is responsible for triggering involuntary (e.g., autonomic and shivering) thermoregulatory responses. Spinal lamina I skin cooling-responsive neurons provide a glutamatergic excitation to neurons in the external lateral subdivision of the lateral parabrachial nucleus (LPBel), which, in turn, project principally to the MnPO of the POA72,73, while a parallel, spinoparabrachial glutamatergic pathway excites POA-projecting neurons in the dorsal subnucleus of the LPB (LPBd)72,74 in response to skin warming74. Thus, activations of POA-projecting LPBd and LPBel neurons, driven respectively by cutaneous, and likely visceral, warm and cold thermoreceptor stimuli, initiate heat defense and cold defense inhibitions and excitations, respectively, in CVC sympathetic outflow and cutaneous blood flow, in BAT sympathetic outflow and BAT thermogenesis, and in shivering EMGs and shivering thermogenesis to maintain a homeostatic Tcore.\n\nSynaptic integration sites throughout the core thermoregulatory network provide the substrate for a wide variety of non-thermal physiological parameters, disease processes, neuromodulators, and drugs to influence the central regulation of Tcore (reviewed in 25). For example, the high metabolic rate of BAT and shivering skeletal muscles during thermogenesis cannot be sustained without a dependable supply of metabolic fuels, particularly oxygen75, lipolytic by-products34, and glucose76. Thus, the CNS networks driving cold-defensive and behavioral BAT activation or shivering are strongly inhibited by signals reflecting a reduction in the short- and long-term availability of these fuel molecules essential for BAT and skeletal muscle metabolism. Similarly, as hypovolemia progresses during dehydration, the ensuing hyperosmolarity reduces the thermoregulatory drive for sweating20,77,78, which serves to prevent cardiovascular collapse79. Some viscerosensory afferents with axons in the vagus nerve and synapsing on second-order neurons in the NTS can also influence BAT activity25,80 and shivering responses and thus are expected to influence the regulation of Tcore. For instance, vagal afferents convey the “metabolic” signals that produce the inhibition of BAT activity induced by upregulation of hepatic glucokinase81 and the BAT activation following either intragastric delivery of the TRP agonist, capsiate82, or the presence of lipids in the duodenum83. Another influence on Tcore includes a prominent hypothermia during motion sickness and nausea84, for which the pathways relating vestibular stimulation to inhibition of CVC and thermogenesis remain to be elaborated.\n\nProviding an additional layer of complexity in the CNS regulation of Tcore is the presence of the thermally responsive TRPV1 channel in the membranes of a variety of classically non-thermal, unmyelinated afferents85–88 that may have access to central thermoregulatory circuits, including via NTS neurons that can inhibit thermogenesis59. The finding that the TRPV1 responsiveness to local brain temperature alters spontaneous glutamate release from the terminals of unmyelinated vagal afferents88,89 provides a potential substrate for Tcore to modulate the activity of second-order sensory neurons in NTS that modulate thermoeffector activation. Importantly, TRPV1 channels are also activated by non-thermal factors, including low (or high) pH, inorganic cations, or endovanilloids, thereby providing a basis for such factors to influence thermoeffector activation and thus Tcore. For instance, TRPV1 channels, potentially on the terminals of afferents in the peritoneum, are stimulated by an endogenous ligand to tonically inhibit BAT thermogenesis, which results in a hyperthermic response to TRPV1 antagonist administration85,90. Although these afferents were not directly tested for their thermal responsiveness, the hyperthermic response to TRPV1 antagonism was not altered by changes in Tcore. The relative influences and the interactions of thermal and non-thermal stimuli on the conductance of the relevant TRPV1 channels could play a role in determining their effect on the level of thermoeffector activation to thermoreceptor stimuli.\n\nInterest in pharmacological modulation of the central thermoregulatory network has focused: (a) on reducing CVC and thermogenesis to lower Tcore; and (b) on augmenting thermogenesis to elevate energy expenditure with the goal of weight loss through consumption of the high-energy lipid stores in white adipose tissue. Novel approaches to reducing Tcore would have immediate benefits in treating intractable fevers that are unresponsive to cyclooxygenase inhibitors. Therapeutic hypothermia can have beneficial effects on survival and on reducing brain and tissue damage in ischemic insults such as cardiac arrest, stroke, and neonatal encephalopathy91–94. Extended space travel (e.g., Mars One) also may require pharmacological induction of a hypothermic, hibernation-like state to reduce energy consumption and psychological stress.\n\nUnder basal metabolic and movement conditions, changes in Tcore must arise from changes in the level of activation of thermoeffector tissues. Although modulating neuronal discharge at any site within the central thermoregulatory network would be expected to alter Tcore, centrally generated hyperthermias, such as fever, that arise from altered activity in hypothalamic thermoregulatory neurons could be most effectively reduced by manipulating thermoeffector efferent pathways95,96. Indeed, directly inhibiting the discharge of neurons in the rRPa area, including the functionally significant premotor neurons that control the principal thermoeffectors, produced a fall in brain temperature of approximately 14°C in an ambient environment of 15°C97, and stimulation of α2 adrenergic receptors in the rRPa could completely block or prevent a lipopolysaccharide-evoked fever95. Similarly, central administration of an adenosine A1 receptor (A1AR) agonist reduced rat Tcore by 10°C in an ambient temperature of 15°C60 and mouse Tcore by 5°C in an ambient temperature of 4°C98. Of particular interest, in rats, this hypothermia, which was produced by a blockade of the cold-evoked activation of BAT and shivering thermogenesis, was long-lasting and paralleled by marked reductions in heart and respiratory rate, in EEG, and in behavior60—all reminiscent of those occurring in hibernation and torpor, which require central stimulation of the A1AR99–101. The discovery of a role for TRP channels in thermal sensation and in thermoeffector activation has stimulated research into the pharmacological modulation of TRP channels in the central thermoregulatory network to abrogate the normal cold defense mechanisms and allow Tcore to fall in a cool ambient environment. By reducing the activation of cooling-responsive skin thermoreceptors, a TRPM8 antagonist reduced rat Tcore by approximately 1°C when the rats were in an ambient temperature of 19°C63. Stimulating TRPV1 reduced mouse Tcore by approximately 12°C when mice were exposed to a 10°C ambient temperature102, and this effect was potentiated by the addition of a TRPM8 antagonist103. The location of the relevant, hypothermic TRPV1 channels remains unknown.\n\nNot only is BAT a thermogenic thermoeffector, including in adult humans104–107, but through its consumption of lipid and glucose energy stores and oxygen, thermogenic metabolism in BAT is a neurally regulated contributor to energy homeostasis. Thus, particularly in the face of an elevated consumption of energy-rich food (e.g., a high-fat diet), a chronic reduction in cooling-evoked BAT thermogenesis would contribute to the augmented adipose energy stores that characterize obesity. Indeed, mice without BAT exhibit a propensity for obesity and diabetes108,109; conversely, overexpression of uncoupling protein-1 (UCP-1), principally responsible for thermogenesis in BAT, mitigates obesity induced by a high-fat diet110. Several anti-obesity therapies currently being explored are based on increased activation of BAT thermogenesis, through either activation of the central thermoregulatory network to increase the sympathetic outflow to BAT111 or an alteration in the cellular biochemical pathways in brown adipocytes or a hyperplasia of BAT to augment thermogenesis. The consistent findings that obese humans have significantly reduced cooling-activated BAT104–106,112, and that the basal (i.e., principally cooling-evoked) sympathetic activation of BAT is reduced in rats fed a high-fat diet113,114, and that a vagal afferent input to the NTS mediates the reduced cooling-evoked BAT activity in rats fed a high-fat diet115 not only support a role for reduced BAT activity in the excess adipose accumulation of obesity but also highlight the significance of non-thermal inputs to the central thermoregulatory network25,81,83 in influencing even the most basic thermoregulatory responses.\n\nConsiderable progress has been achieved in revealing the functional organization of the dedicated thermoregulatory network within the CNS that provides the fundamental neural control of the thermoregulatory effectors: thermoregulatory behavior, CVC, and BAT and shivering thermogenesis, although many of the details of the neurophysiology and neuroanatomy of the central thermoregulatory network remain active areas of investigation. The changes in Tcore that accompany a wide range of behaviors and in response to many hormones and drugs arise through altered non-thermal inputs to, or neurochemical modulation of, the neural activity within the fundamental thermoregulatory network. The latter, as well as thermoreceptor-based strategies, are being researched as therapeutic approaches in which the central thermoregulatory networks are recruited to alter Tcore and metabolism.",
"appendix": "Competing interests\n\n\n\nThe author declares that he has no competing interests.\n\n\nGrant information\n\nThis work was supported by National Institutes of Health grants R01NS040987 and R01NS091066 to Shaun F. Morrison.\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\nOotsuka Y, de Menezes RC, Zaretsky DV, et al.: Brown adipose tissue thermogenesis heats brain and body as part of the brain-coordinated ultradian basic rest-activity cycle. Neuroscience. 2009; 164(2): 849–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVujovic N, Gooley JJ, Jhou TC, et al.: Projections from the subparaventricular zone define four channels of output from the circadian timing system. J Comp Neurol. 2015; 523(18): 2714–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRomanovsky AA: Do fever and anapyrexia exist? Analysis of set point-based definitions. 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PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nNakamura K, Morrison SF: A thermosensory pathway mediating heat-defense responses. Proc Natl Acad Sci U S A. 2010; 107(19): 8848–53. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMadden CJ, Morrison SF: Hypoxic activation of arterial chemoreceptors inhibits sympathetic outflow to brown adipose tissue in rats. J Physiol. 2005; 566(Pt 2): 559–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMadden CJ: Glucoprivation in the ventrolateral medulla decreases brown adipose tissue sympathetic nerve activity by decreasing the activity of neurons in raphe pallidus. Am J Physiol Regul Integr Comp Physiol. 2012; 302(2): R224–32. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBaker MA, Doris PA: Control of evaporative heat loss during changes in plasma osmolality in the cat. J Physiol. 1982; 328(1): 535–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTakamata A, Mack GW, Gillen CM, et al.: Osmoregulatory modulation of thermal sweating in humans: reflex effects of drinking. Am J Physiol. 1995; 268(2 Pt 2): R414–22. PubMed Abstract\n\nKenney WL, Stanhewicz AE, Bruning RS, et al.: Blood pressure regulation III: what happens when one system must serve two masters: temperature and pressure regulation? Eur J Appl Physiol. 2014; 114(3): 467–79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSzékely M: The vagus nerve in thermoregulation and energy metabolism. Auton Neurosci. 2000; 85(1–3): 26–38. PubMed Abstract | Publisher Full Text\n\nTsukita S, Yamada T, Uno K, et al.: Hepatic glucokinase modulates obesity predisposition by regulating BAT thermogenesis via neural signals. Cell Metab. 2012; 16(6): 825–32. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nOno K, Tsukamoto-Yasui M, Hara-Kimura Y, et al.: Intragastric administration of capsiate, a transient receptor potential channel agonist, triggers thermogenic sympathetic responses. J Appl Physiol (1985). 2011; 110(3): 789–98. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBlouet C, Schwartz GJ: Duodenal lipid sensing activates vagal afferents to regulate non-shivering brown fat thermogenesis in rats. PLoS One. 2012; 7(12): e51898. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nNgampramuan S, Cerri M, Del Vecchio F, et al.: Thermoregulatory correlates of nausea in rats and musk shrews. Oncotarget. 2014; 5(6): 1565–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRomanovsky AA, Almeida MC, Garami A, et al.: The transient receptor potential vanilloid-1 channel in thermoregulation: a thermosensor it is not. Pharmacol Rev. 2009; 61(3): 228–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeters JH, McDougall SJ, Fawley JA, et al.: Primary afferent activation of thermosensitive TRPV1 triggers asynchronous glutamate release at central neurons. Neuron. 2010; 65(5): 657–69. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nFawley JA, Hofmann ME, Andresen MC: Cannabinoid 1 and transient receptor potential vanilloid 1 receptors discretely modulate evoked glutamate separately from spontaneous glutamate transmission. J Neurosci. 2014; 34(24): 8324–32. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nFawley JA, Hofmann ME, Largent-Milnes TM, et al.: Temperature differentially facilitates spontaneous but not evoked glutamate release from cranial visceral primary afferents. PLoS One. 2015; 10(5): e0127764. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nShoudai K, Peters JH, McDougall SJ, et al.: Thermally active TRPV1 tonically drives central spontaneous glutamate release. J Neurosci. 2010; 30(43): 14470–5. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSteiner AA, Turek VF, Almeida MC, et al.: Nonthermal activation of transient receptor potential vanilloid-1 channels in abdominal viscera tonically inhibits autonomic cold-defense effectors. J Neurosci. 2007; 27(28): 7459–68. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHypothermia after Cardiac Arrest Study Group: Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. N Engl J Med. 2002; 346(8): 549–56. 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PubMed Abstract | Publisher Full Text\n\nLevin BE: Reduced norepinephrine turnover in organs and brains of obesity-prone rats. Am J Physiol. 1995; 268(2 Pt 2): R389–94. PubMed Abstract\n\nMadden CJ: Consumption of a high fat diet inhibits sympathetic outflow to brown adipose tissue (BAT) via vagal afferent activation of neurons in the Nucleus Tractus Solitarius (NTS). Auton Neurosci. 2015; 192: 13. Publisher Full Text"
}
|
[
{
"id": "13787",
"date": "12 May 2016",
"name": "Robin M McAllen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13818",
"date": "12 May 2016",
"name": "Matteo Cerri",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13819",
"date": "12 May 2016",
"name": "Andrej A Romanovsky",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-880
|
https://f1000research.com/articles/5-879/v1
|
12 May 16
|
{
"type": "Review",
"title": "Molecular pathogenesis of hepatocellular carcinoma and impact of therapeutic advances",
"authors": [
"Renumathy Dhanasekaran",
"Salome Bandoh",
"Lewis R. Roberts",
"Renumathy Dhanasekaran",
"Salome Bandoh"
],
"abstract": "Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality and has an increasing incidence worldwide. HCC can be induced by multiple etiologies, is influenced by many risk factors, and has a complex pathogenesis. Furthermore, HCCs exhibit substantial heterogeneity, which compounds the difficulties in developing effective therapies against this highly lethal cancer. With advances in cancer biology and molecular and genetic profiling, a number of different mechanisms involved in the development and progression of HCC have been identified. Despite the advances in this area, the molecular pathogenesis of hepatocellular carcinoma is still not completely understood. This review aims to elaborate our current understanding of the most relevant genetic alterations and molecular pathways involved in the development and progression of HCC, and anticipate the potential impact of future advances on therapeutic drug development.",
"keywords": [
"hepatocellular carcinoma",
"liver cancer",
"HCC"
],
"content": "Introduction\n\nHepatocellular carcinoma (HCC) is the most common primary liver malignancy and the sixth most common cancer worldwide1. It is an aggressive malignancy with a poor prognosis and is currently the second most common cause of cancer-related mortality. Although more than 80% of the estimated 782,000 new cases of HCC in 2012 occurred in less developed regions of the world, its incidence is increasing worldwide, including in more developed countries1.\n\nThe most common risk factors for HCC development are chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, and the prevalence of HCC mirrors the occurrence of these infections2,3. Other major risk factors include alcoholic cirrhosis, non-alcoholic steatohepatitis (NASH), consumption of aflatoxin-contaminated foods, and exposure to other chemical carcinogens4. Heavy alcohol use increases the risk for HCC and also has been reported to have synergistic effects with other risk factors such as obesity and viral hepatitis5,6. NASH is the most rapidly growing indication for liver transplantation for patients with HCC in the US, and the annual incidence of HCC in patients with NASH cirrhosis is 2.6%7,8. There is more recent recognition that the metabolic syndrome and its components such as diabetes and obesity also increase the risk for HCC9–11. Pre-existing metabolic syndrome has been shown to confer a 2.1-fold increased risk for HCC which is independent of other risk factors12. In this review, we describe the pathogenic mechanisms by which these diverse etiologic factors interact with the molecular milieu in the liver to drive the oncogenesis of HCC.\n\n\nNatural history of precancerous lesions\n\nMost causes of HCC mediate liver injury through the development of liver inflammation and fibrosis, which eventually results in the disordered liver architecture characteristic of liver cirrhosis; thus, cirrhosis precedes HCC in 80–90% of patients. Cirrhotic livers exhibit focal areas of abnormal, immature hepatocytes and these dysplastic foci (<1 mm) or dysplastic nodules (DNs) (≥1 mm) arising in the background of cirrhosis are considered precancerous lesions. DNs are classified into low and high grade on the basis of the presence of atypia and other morphologic features. Although both low- and high-grade nodules have the potential to evolve into HCC, high-grade DN has a much greater risk. Differentiating DNs, especially high-grade DN, from early HCC can be challenging, and an international consensus guideline provides recommendations for making this distinction13. The presence of stromal invasion is considered to be the hallmark feature that differentiates early HCC from DNs. HCC is also subclassified into early HCC and progressed HCC, with differing long-term clinical outcomes13. Early HCC refers to small (<2 cm) well-differentiated (grade 1) tumor nodules with indistinct margins and this is now accepted as a separate entity with a good prognosis. Figure 1 depicts the proposed natural history and typical features of precancerous lesions and HCC.\n\nDysplastic foci (<1 mm) or dysplastic nodules (DNs) (≥1 mm) are considered precancerous lesions and are classified into low and high grade. Both low- and high-grade nodules have the potential to evolve into HCC, but high-grade DN have a much greater risk. Early HCCs are characterized by the presence of stromal invasion. HCC is further subclassified into early HCC and progressed HCC based on features noted in the figure. Abbreviations: HCC, hepatocellular carcinoma; HG-DN, high-grade dysplastic nodule; LG-DN, low-grade dysplastic nodule.\n\n\nHistologic classification of hepatocellular carcinoma\n\nHCC is clinically heterogeneous and interestingly the histopathologic appearance of HCC also exhibits significant heterogeneity. The range of cellular differentiation extends from very well-differentiated to poorly differentiated tumors. HCCs also exhibit varied morphologic subtypes, including biphenotypic HCCs with combined features of hepatocellular and cholangiocarcinoma (4–5%), cirrhotomimetic HCC, clear cell HCC, fibrolamellar HCC, granulocyte colony-stimulating factor HCC with major neutrophilic infiltrates, lymphocyte-rich HCC, myxoid HCC, sarcomatoid HCC, scirrhous HCC, and steatohepatitic HCC. Despite the obvious histological diversity of HCCs, there has been only limited correlation of histological features with known molecular or genetic aberrations in HCC and almost no developed capability to translate histological or molecular characteristics to the selection of optimal therapies. Contemporary efforts to correlate histological subtypes of HCC with molecular features are beginning to yield fruit and improve our understanding of HCC pathogenesis and genotype-phenotype correlations. The recent discovery that a chimeric fusion protein involving protein kinase A is present in 100% (15 out of 15) of fibrolamellar HCCs is one such example14,15. The chromophobe morphologic subtype of HCC has also been reported to exhibit alternative lengthening of telomeres (ALT), a specific molecular mechanism to overcome replicative senescence16.\n\n\nGenetic and epigenetic changes in the molecular carcinogenesis of hepatocellular carcinoma\n\nNew advances in next-generation sequencing have yielded significant insights into the genomic landscape of HCC. Several recent studies have explored various aspects of HCC by using whole genome sequencing, whole exome sequencing, RNA sequencing, and genome-wide methylation assays. Results thus far describe a complex and heterogeneous malignancy exhibiting a wide array of genetic and epigenetic changes. Below, we discuss some of the pertinent alterations that play a role in the pathogenesis of HCC.\n\nRecurrent somatic mutations in specific genes are well recognized as potential drivers of carcinogenesis. Melanoma and lung cancer have the highest rate of mutations per genome, whereas HCCs usually have an intermediate number of mutations per genome similar to other solid tumors (typically 20–100 mutations per genome)17. The underlying etiology of liver cancer also appears to influence the occurrence of specific mutations. For example, HBV is associated with a relatively high frequency of mutations as it replicates through RNA-mediated reverse transcription, and the HBV reverse transcriptase (HBV RT) does not have proofreading function. In contrast, HCV is a single-stranded non-retroviral RNA virus that, unlike HBV, does not integrate into the host genome. However, HCV can cause double-stranded DNA breaks and increase the mutation frequency. HCV-infected cells exhibit an increased mutation frequency in genes such as the immunoglobulin genes, BCL-6, TP53, and β-catenin (CTNNB1)18. Most recently, HCCs have been observed that have a hypermutated genotype with a mutational spectrum characteristic of that caused by the herbal mutagen aristolochic acid, the toxic ingredient of the Chinese herbal preparation wild ginger19. Below, we discuss some of the commonly observed somatic mutations in HCC.\n\nTelomerase promoter mutations. Telomeres are located at the tips of linear chromosomes and function to protect the chromosome from end-to-end fusion and destruction by nucleases or ligases or both. Telomerase is an enzymatic protein complex made up of the telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC). Telomerase maintains telomere length by synthesizing specific telomeric DNA sequences and adding them to the end of the chromosome. Telomerase expression is usually suppressed in mature adult cells. Thus, DNA polymerase is unable to fully replicate the terminal chromosomal segment and telomeres become progressively shorter with repeated cell divisions. In chronic liver injury where there is high cell turnover, telomere shortening is accentuated. Telomere shortening beyond a certain critical length leads to activation of a DNA damage program which results in apoptosis or cellular senescence that results in the inability of the liver to fully regenerate a normal architecture, triggering the development of liver fibrosis and, eventually, cirrhosis. The telomere-shortening effect of chronic liver injury can synergize with inherited genetic variants in the TERT and TERC genes that result in decreased activity of the telomerase complex to accelerate the premature development of liver fibrosis and cirrhosis20,21. Because cirrhosis is a precursor to HCC, the telomere hypothesis holds that this telomere shortening results in chromosomal instability that drives cancer initiation. Stabilization of the telomeric DNA through either increased telomerase expression or alternative mechanisms of telomerase activation is a key mechanism of cellular immortalization, allowing cells to survive and proliferate indefinitely22. Mutations in the TERT promoter region have now been shown to be the most common mutation in HCC and the most frequent mechanism of telomerase activation. The mutations result in the formation of novel ETS transcription factor-binding sites upstream of the TERT start site, which leads to increased TERT transcript expression. Mutations in the TERT promoter region occur in 30–60% of HCCs23–26. Nault et al. found TERT promoter mutations not only in 59% of HCCs but also in 25% of cirrhotic preneoplastic lesions, suggesting that this is likely a driver mutation24. Interestingly, TERT promoter mutations are conspicuously less common in HBV-induced HCCs, but these tumors have been shown to have recurrent integrations of HBV sequences into the TERT gene locus, which serves as a complementary mechanism for telomerase activation27–29.\n\nTP53 pathway mutations. TP53 is a widely recognized tumor suppressor, and low p53 levels or mutations in p53 are found in multiple cancer types. Wild-type p53 promotes apoptosis and cell cycle arrest, therefore, inactivating mutations in the p53 gene, or other pathway components, may render hepatocytes susceptible to the effects of other carcinogens that activate oncogenic pathways and may also predispose to the development of HCCs with a more aggressive phenotype30. The frequency of p53 gene mutation in HCCs ranges from 18% to 50%, depending on the underlying etiology. Consequently the rate of p53 mutations varies in different geographic regions, reflecting the regional variations in HCC etiology26,29,31–33. In particular, dietary exposure to fungal aflatoxin (AFB1) results in a specific p53 mutation most commonly reported at codon 249; this is considered to be a driver mutation since it is also found in the normal livers of patients exposed to AFB134. There is strong epidemiologic synergism between aflatoxin exposure and chronic HBV infection in the induction of HCC, and it has been shown that in patients infected with hepatitis B, expression of hepatitis B X (HBx) is associated with an approximately twofold increase in the incidence of G/C-to-T/A transversion mutations following AFB1 exposure35. Other genes in the p53 pathway that are recurrently mutated in HCC include ATM and RPS6KA329.\n\nOther common somatic mutations and hepatitis B virus integrations in hepatocellular carcinoma. Some of the other common mutations in HCC involve the Wnt/β-catenin pathway, including mutations in the β-catenin (CTNNB1) (18–40%), AXIN1 and AXIN2 genes31–33. These, along with other alterations in Wnt/β-catenin pathway components, are discussed below. Additional recently identified mutations in HCCs involve members of the chromatin remodeling pathway (ARID1A and ARID2) and the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway (JAK1, IL6R, and IL6ST), genes involved in ubiquitination (KEAP1), genes involved in RAS/MAPK signaling (RPS6KA3) and genes in the oxidative stress pathway (NFE2L2)36.\n\nHBV is known to recurrently integrate into the host genome and promote hepatocarcinogenesis37. Several studies of the sites of HBV integrations in the hepatocyte genome have identified genes recurrently targeted by HBV integration, including TERT, MLL4, RARβ, CCNE1, Cyclin A2, FN1, ROCK1, SENP5, ANGPT1, platelet-derived growth factor (PDGF) receptor, calcium signaling-related genes, ribosomal protein genes, epidermal growth factor receptor (EGFR), and mevalonate kinase carboxypeptidase28,37–40. Several of these integrations are proposed to have direct or indirect pathogenic roles in the development for HCC. For example, MLL4 encodes a histone methyltransferase that plays an important role in epigenetic modification of gene expression41. Also, HBV integration into specific genes has been noted to alter gene expression; for example, Sung et al. reported that samples with HBV integration had significantly higher expression of the TERT, MLL4, and CCNE1 genes than tumors not harboring HBV DNA integrations in these genomic regions39. Moreover, HBV viral integration also results in deletions or translocations of the host genome and ultimately increases chromosomal instability, which also predisposes to cancer initiation42–44.\n\nCopy number variations are structural alterations of the genome in which small or large segments of the chromosome are either amplified (gain of genomic DNA) or deleted (loss of genomic DNA). Such structural variations promote carcinogenesis by increased expression or activation of oncogenes and decreased expression or inactivation of tumor suppressors. A recent study of 125 HCCs reported focal amplifications in 32% of the HCCs, identifying CCND1 and FGF19 as genes recurrently amplified in HCC32. Focal deletions appeared to be even more prevalent, being present in 40% of HCCs; deletions commonly involved the CDKN2A (encoding the p16 tumor suppressor), CDKN2B, AXIN1, and IRF2 genes32. Another study, of 286 HCC patients, identified 29 recurrently amplified regions and 22 recurrently deleted regions with a high level of copy number changes. Genes commonly involved with copy number variations included CCND1, MET, CDKN2A, and CDKN2B45. Other studies of smaller sample cohorts similarly reported regions exhibiting significant copy number variations and used this analysis as a strategy for identifying potential HCC driver genes46,47. Several of these alterations have known associated pathogenic mechanisms; for example, a proportion of HCCs activate TERT by focal amplification in the TERT region, and deletion of AXIN1 is one of the mechanisms mediating Wnt/β-catenin pathway activation in HCC.\n\nAnother form of somatic variation contributing to carcinogenesis is chromosomal rearrangements, which can result in the fusion of two genes by chromosome translocation, inversion, or deletion. In a recent breakthrough study, such a gene fusion was described in fibrolamellar HCC. This is a variant of HCC that arises in non-cirrhotic livers, usually in young persons, and has a distinct morphology. A chromosomal rearrangement involving an approximately 400-kilobase deletion in chromosome 19 results in the formation of a chimeric RNA encoding a protein containing the amino-terminal domain of DNAJB1 (a homolog of the molecular chaperone DNAJ), fused in frame with PRKACA (the catalytic domain of protein kinase A). This fusion appears to be highly specific for fibrolamellar HCCs, being identified in 100% of fibrolamellar HCCs, suggesting that this genetic alteration likely contributes to tumorigenesis and the unique morphology of this HCC subtype14,15.\n\nEpigenetics is defined by the presence of heritable states of gene expression without alteration in DNA sequences. Deregulated epigenetics contributes to carcinogenesis by influencing multiple mechanisms, including gene transcription, chromosomal stability, and cell differentiation. Epigenetic mechanisms include changes in the methylation, hydroxymethylation48, or acetylation (or a combination of these) of particular DNA regions or of the histone proteins around which DNA is organized, as well as mechanisms of gene regulation by non-coding RNAs.\n\nDNA methylation. Dysregulated methylation targets multiple gene regions in HCC and is characterized by global and site-specific hypomethylation as well as site-specific hypermethylation. Global hypomethylation in liver cancer affects the structural-nuclear function by promoting chromosomal and genetic instability, whereas regional hypermethylation is often associated with silencing of tumor suppressor genes49. Etiological factors such as chronic HBV and HCV infection may cause dysregulated methylation during liver carcinogenesis50,51. Deng et al. reported on a subset of 15 genes that were found to be preferentially methylated in HCV-related HCC; the methylated genes belong to signaling pathways such as RAS/RAF/ERK and the Wnt/β-catenin pathways52. Methylation of the GSTP1 and E-cadherin promoters has been reported to preferentially occur in hepatitis B-related HCC53. Another epigenetic mechanism for tumorigenesis by HBV is targeted deregulation of DNA methyltransferases (DNMTs) by HBx, which promotes both specific regional hypermethylation and global hypomethylation50.\n\nCDKNA2 promoter hypermethylation leading to suppression of p16 is a commonly observed event in HCC54,55. P16 is a cell cycle regulator and a tumor suppressor; hence, its suppression promotes tumor progression. Other commonly methylated genes in HCC include RASSF1A56, GSTP157,58, SOCS-359, and MGMT60. More recently, whole genome approaches to characterizing changes in methylation have resulted in more comprehensive assessments of gene methylation in cancer and allowed integration of whole genome methylation with whole genome gene expression data, identifying genes whose expression is truly modulated by methylation61–63.\n\nHistone modification. Histones regulate gene expression by determining the open or closed state of chromatin; thus, the level of gene expression depends on the post-translational modifications of histones in the transcriptional unit. Post-translational histone modifications such as acetylation and methylation of lysine and arginine residues, phosphorylation of serine and threonine residues, and ubiquitination of lysines are directed at the histone tails that protrude from the nucleosomes. The role of such DNA-protein modifications in liver carcinogenesis and HCC progression is not fully understood. High levels of trimethylated histone H3 lysine 4 (H3K4me27) have been shown to correlate with reduced overall survival and poor prognosis in HCC64. In another study, high levels of H3K27me3 correlated with aggressive tumor features such as vascular invasion, large tumor size, multiplicity of tumors, and poor differentiation, and predicted worse prognosis in HCC65. The regulation of histone modifications appears to be specific to different etiologies. For example, in patients with HBV, the oncogenic HBx protein can interact directly with the CBP/p300 histone acetyl-transferase complex, thus altering gene transcription and promoting tumorigenesis66.\n\nChromatin remodeling. Chromatin remodeling describes the process of dynamic changes in chromatin structure that regulate gene expression, apoptosis, and DNA repair. Disruption in chromatin remodeling can contribute to cancer initiation and progression. Awareness of the influence of chromatin remodeling processes in HCC development and growth is increasing. For example, the switch/sucrose non-fermenting (SWI/SNF) complex is a multi-protein complex essential for chromatin remodeling. SWI/SNF comprises dozens of proteins, including SMARCB1 and SMARCA4, and plays a key role in epigenetic regulation of gene expression. Recent studies have described frequent mutations in SW1/SNF chromatin remodeling complex genes such as ARID1A, ARID1B, and ARID2 in HCC67. ARID2, which encodes a SW1/SNF regulatory subunit protein, was found to be mutated in 18.5% of HCV-related HCCs68. Most ARID1A and ARID2 mutations detected in cancer cells to date are inactivating mutations, suggesting that both proteins function as tumor suppressors.\n\nThe polycomb group of chromatin remodeling proteins also plays a role in heritable gene silencing. There are two polycomb repressive complexes, denoted PRC1 and PRC2. The mechanisms by which they repress gene expression are incompletely understood, but PRC1 is believed to work through ubiquitin ligases which covalently modify histone tails, whereas the main function of PRC2 is to methylate histone H3K2769,70. EZH2, a subunit of PRC2, is a methyltransferase that mediates gene silencing71. EZH2 mRNA transcript and protein levels are consistently elevated in HCC in comparison with non-tumor liver tissues, and high levels of EZH2 are associated with HCC invasion and metastasis and poor prognosis72,73. EZH2 has been shown to promote hepatocarcinogenesis by silencing Wnt antagonists and consequently activating Wnt/β-catenin signaling72. Knockdown of EZH2 in liver cancer cell lines also reduces levels of the repressive H3K27me3 histone, resulting in re-expression of a distinct subpopulation of tumor suppressor miRNAs that control cell motility and adhesion74.\n\nMicroRNAs. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the translation of many genes. They have emerged as key factors regulating multiple biological processes, including development, differentiation, and cell proliferation. MiRNAs mediate carcinogenesis and progression of HCC by directly or indirectly controlling the expression of key proteins involved in cancer-associated pathways.\n\nChronic hepatitis and hepatocarcinogenesis are associated with profound changes in miRNA expression75. MiR-155, a positive regulator of liver inflammation, is upregulated in both serum and monocytes of patients with chronic HCV. The HCV core, NS3, and NS5 proteins and the HCV-induced TLR4 and TLR8 ligands all mediate increased miR-155 and tumor necrosis factor alpha (TNFα) production in chronic HCV infection, and this in turn promotes hepatocarcinogenesis by activating Wnt signaling76. MiR-122, one of the most abundant liver-specific miRNAs, is downregulated in about 70% of HCCs77. MiR-122 acts as a tumor suppressor, inducing apoptosis of HCC cells by directly targeting the Wnt/β-catenin pathway. Suppression of miR-122 is associated with intrahepatic metastases and tumor recurrence after surgical resection78–80. In a phenomenon reflecting the complexity of cancer genetic mechanisms, deletions in miR-122a have been shown to promote the epithelial-mesenchymal transition (EMT) and spontaneous HCC formation in mouse models81. Other miRNAs such as miR-224, miR-224-3p, and their precursors are upregulated in HCV-associated cirrhosis, HCV-associated HCC, and HBV-associated liver failure compared with normal liver tissue82. Apoptosis inhibitor 5 (API-5) and SMAD4 have been identified as target genes for miR-224, and expression of miR-224 is associated with poor survival83. Overexpression of miR-224 increases SMAD4 protein in murine granulosa cells without increasing SMAD4 RNA levels, suggesting a post-transcriptional role for miR-22483. Tumor suppressive miRNAs, including miR-1, miR-124, miR-214, miR-34-A, and miR-449, target mRNAs involved in cell growth, metastasis, or suppression of apoptosis and usually are downregulated in HCC. In contrast, oncogenic miRNAs, including miR-221, miR-224, miR-21, and miR210, promote tumor progression and are upregulated in HCCs84. There are active efforts under way to exploit modulation of miRNA levels for HCC therapy85.\n\nLong non-coding RNAs. Long non-coding RNAs (lncRNAs) are another class of transcribed RNAs that do not encode proteins. LncRNAs regulate gene expression and protein synthesis by diverse mechanisms. Aberrant expression of lncRNA can affect genes involved in hepatocarcinogenesis, microvascular invasion, and metastasis. Most lncRNAs are undetectable or expressed at low levels in normal liver tissue but upregulated in HCCs. Alterations in several lncRNAs have been described in HCC. In HBV-related HCC, HBx induces upregulation of an lncRNA known as highly upregulated in liver cancer (HULC), which in turn suppresses the expression of p18 and facilitates proliferation of HCC86. Depletion of HULC results in significant deregulation of several genes involved in liver cancer, and higher HULC levels are observed in the plasma of patients with higher histological grades of HCC or positive HBV status87. HULC is specifically increased in blood and tumor tissues of patients with HCC and may have utility as a biomarker for HCC. Another lncRNA, designated lncRNA-HEIH, has also been shown to be overexpressed in HCC88. Downregulation of lncRNA-HEIH induced G0/G1 cell cycle arrest. The level of overexpression of lncRNA-HEIH was associated with recurrence of tumor in HBV-related HCC and was also an independent risk factor for survival88. Lnc-RNA-Dreh, also known as the HBx-related lncRNA, acts as a tumor suppressor by targeting the intermediate filament protein vimentin in HBV-associated HCC, leading to inhibition of growth and metastases both in vitro and in vivo89. The novel lncRNA-associated microvascular invasion in HCC (lnc-MVIH) has been found to promote tumor growth and intrahepatic metastasis through activation of angiogenesis90. It has been shown that lnc-MVIH inhibits secretion of phosphoglycerate kinase 1 (PGK1), a glycolytic enzyme known to inhibit angiogenesis90,91. The lncRNA HOX transcript antisense RNA (HOTAIR), which can reprogram chromatin to promote cancer metastasis, has been found to be overexpressed in HCC, and patients with HOTAIR overexpression had significantly poorer prognosis and higher recurrence rates than those with low HOTAIR expression92,93. Mineral dust-induced gene (MDIG), an lncRNA regulated by the c-myc oncogene, was also found to be overexpressed in HCC. MDIG expression was noted in the nuclei of neoplastic cells and had higher expression in larger and poorly differentiated HCCs94.\n\nAnother mechanism by which lncRNAs induce hepatocarcinogenesis was recently described by Lau et al., who demonstrated that HBV viral integrations into the host genome can result in viral-human chimeras which function as lncRNAs and can promote tumorigenicity95. An HBV integration in chromosome 8 resulted in co-transcription of long-interspersed nuclear element 1 (LINE1), which is usually silent in the human genome, with the HBx gene of HBV. They were further able to demonstrate that the HBx-LINE1 chimera was functionally relevant as it led to Wnt/β-catenin pathway activation and resulted in tumor progression. Other recently described lncRNAs which play a role in hepatocarcinogenesis and also have the potential to serve as biomarkers include MALAT96, HOTTIP97, and MEG398.\n\n\nKey signaling pathways in liver carcinogenesis\n\nAlterations in numerous signaling pathways occur in cancer, and several specific pathways have been observed to be dysregulated in HCC. Changes in liver tissues induced either by chronic viral infection or by exposure to hepatotoxic agents cause upregulation of components of a number of cellular signaling pathways. The predominant pathways involved in HCC pathogenesis include pathways regulating growth factor signaling such as the insulin-like growth factor (IGF), epidermal growth factor (EGF), PDGF, fibroblast growth factor (FGF) and hepatocyte growth factor (HGF/MET); pathways related to cell differentiation such as the WNT, Hedgehog, and Notch pathways; and pathways related to angiogenesis such as the vascular endothelial growth factor (VEGF) and FGF pathways. The major signaling mediators downstream of the receptor tyrosine kinases are the Ras/Raf/MEK/ERK and P13K/AKT/mTOR cascades99. There are also substantial contributions to liver carcinogenesis from pathways regulating the tumor microenvironment and pathways that disrupt anti-tumor immunity88,100. Below, we describe the most commonly altered pathways and the mechanisms that lead to specific pathway activation. Besides improving our understanding of the pathogenesis of HCC, this information is valuable for the identification of novel drug targets.\n\nThe WNT/β-catenin signaling pathway is implicated in embryogenesis, differentiation, cell proliferation, and tumorigenesis and is one of the most commonly disrupted pathways in HCC101. Nineteen Wnt ligands have been described and there are 10 transmembrane frizzled receptors to which they bind, leading to either canonical (β-catenin-dependent) or non-canonical (β-catenin-independent) Wnt pathway activation. Activation of the canonical Wnt signaling pathway results in accumulation of β-catenin in the cytoplasm and translocation to the nucleus, where it binds to transcription factor TCF/LEF and activates downstream target genes. Gene mutations that activate WNT/β-catenin signaling are seen in up to 50% of HCCs. The most common are activating mutations in CTNNB1, which result in stabilization of β-catenin32. Additionally, mutations in AXIN1 (in 3–16% of HCCs) and AXIN 2 (in about 3% of HCCs) which are both negative regulators of the Wnt pathway, as well as inactivation of the tumor suppressor gene adenomatous polyposis coli (APC), contribute to Wnt pathway activation32. There are multiple additional mechanisms of Wnt pathway activation. In fact, those HCCs that exhibit Wnt/β-catenin pathway activation without CTNNB1 mutation appear to be distinct from those with CTNNB1 mutations. HCCs with Wnt/β-catenin pathway activation without CTNNB1 mutation are preferentially seen in HBV-infected patients and are associated with high chromosomal instability and an aggressive phenotype102. In contrast, HCCs with CTNNB1 mutation are usually low-grade tumors with good prognosis103,104.\n\nThere are several other mechanisms for activation of Wnt signaling in HCCs apart from mutations involving the pathway. A subset of HCCs with Wnt pathway activation in the absence of CTNNB1 mutation show evidence of crosstalk with the transforming growth factor-beta (TGFβ) pathway102. Overexpression of Wnt ligands105 or frizzled receptors106 and epigenetic changes in secreted frizzled-related protein-1107 are other mechanisms by which the Wnt pathway is activated in HCC.\n\nActivation of receptor tyrosine kinases induce the Ras-mitogen-activated protein kinase (MAPK or extracellular signaling regulated kinase, or ERK) and phosphatidylinositol 3-kinase (PI3K)-Akt kinase signaling pathways in about 50% of HCCs108. Ligand binding and phosphorylation of several growth factor tyrosine kinase receptors, including the EGFR, FGFR, HGFR/c-MET, the stem cell growth factor receptor (c-kit), and VEGFR, lead to activation of the MAPK and PI3K pathways. Ras/Raf/MEK/ERK (MAPK) pathway activation in turn activates proto-oncogene cFos and transcription factor AP-1/c-Jun, which induce transcription of genes that drive cell proliferation109. Activation of the PI3K-Akt kinase signaling pathway through the insulin or IGF receptors (such as IGFR1) results in disruption of the mammalian target of rapamycin (mTOR) pathway, which occurs in about 40% to 50% of cases of HCC, thus promoting carcinogenesis110. This pathway can also be dysregulated by constitutive activation of PI3K because of loss of function of the tumor suppressor gene PTEN by either mutation or epigenetic silencing. Sorafenib, which is currently the only approved therapy for advanced HCC, acts in part by blocking the RAS/MEK/ERK pathway111. Although the role of EGFR mutations in HCC pathogenesis is small, the EGFR pathway appears to play a significant role in HCC initiation, as a polymorphism in the EGF gene (SNP rs44449030) (G/G versus A/A) was associated with a fourfold increased risk for HCC in patients with cirrhosis112. Another receptor tyrosine kinase pathway that has garnered increased attention recently is the HGF-MET pathway. Expression of a MET gene signature was associated with vascular invasion and poor prognosis in human HCC, and in a subgroup analysis of the Sorafenib HCC Assessment Randomized Protocol (SHARP) trial, high plasma HGF levels were found to correlate with poor survival in patients who received sorafenib113,114. The FGF family consists of 23 members whose multiple ligands interact with four FGF receptors (FGFR1-4), of which FGFR4 is the most abundant receptor expressed in hepatocytes. The ligand FGF19 binds to FGFR4 and regulates bile acid synthesis and hepatocyte proliferation. Recent data have shown that FGF19-FGFR4 pathway activation may play a key role in a proportion of HCCs and this pathway is a potential therapeutic target115,116. A new small molecule-specific inhibitor of FGFR4 has been shown to be efficacious against HCCs with an intact FGF pathway117.\n\nA number of receptor tyrosine kinases use heparan sulfate as a co-receptor, and heparan sulfate on the cell surface or in the extracellular matrix can also serve as a storage or concentration site for heparan sulfate-binding ligands. A pair of heparan sulfate sulfatases, SULF1 and SULF2, have been shown to modulate HCC carcinogenesis and tumorigenesis through effects on the affinity of heparan sulfate for heparan sulfate-binding receptor tyrosine ligands. Besides the efforts to identify specific inhibitors of receptor tyrosine kinases, there are efforts under way to develop sulfatase inhibitors as potential anti-cancer agents118–120.\n\nHCC is a highly vascular tumor and angioneogenesis is a dominant feature of this tumor, with the hepatic artery as the major source of its blood supply. VEGF and angiopoetins play a prominent role in promoting and sustaining neoangiogenesis in HCC121,122. These principles have been used for developing effective therapeutic strategies against HCC, such as transarterial chemoembolization (TACE), which works by blocking the arterial supply to the tumor, and sorafenib, which inhibits the angiogenic effects of growth factors such as VEGF. In spite of the rich vascular supply, hypoxia is present in focal areas of the tumor because of disorganized capillarization and the presence of leaky, immature vessels123. Hypoxia in HCC, in turn, leads to induction of growth factors such as hypoxia-inducible factors 1 and 2 (HIF 1 and 2) and IGFs that promote further tumor angiogenesis by transcriptional activation of hypoxia-responsive genes and lead to tumor progression and metastases124,125. HIFs have also been shown to impart chemo- and radio-resistance to HCC tumors, leading to failure of transarterial therapies126, and hence their overexpression is associated with poor prognosis.\n\nThe TGFβ pathway has long been recognized to play a dual role in cancer: it has the ability to suppress cellular growth in the early stages of cancer initiation and the paradoxical ability to promote invasiveness and angiogenesis in later stages127. With this in mind, Coulouarn et al. used transcriptome analysis to identify early and late TGFβ signatures in HCC and showed that the late TGFβ signature was associated with shortened survival time compared with patients with the early TGFβ signature. Also, tumors expressing late TGFβ-responsive genes displayed an invasive phenotype and increased tumor recurrence128. The poor prognosis observed is likely explained by the observation that, in late stages of HCC, TGFβ is known to promote EMT, which is a key mechanism involved in promoting tumor metastases129. In another widely recognized transcriptomic classification of HCCs, the subgroup S1 was noted to be associated with WNT pathway activation which was the result of TGFβ activation102. These data suggest that TGFβ pathway activation is likely involved in a significant subset of HCCs and hence is a rational drug target130. In recent work from our group, we have shown that the heparan sulfate sulfatases promote HCC tumor progression by activating the TGFβ pathway118,131.\n\nSTATs are activated by a variety of cytokines, hormones, and growth factors. The activation occurs through tyrosine phosphorylation by JAKs. Activated STATs stimulate the transcription of suppressors of cytokine signaling (SOCS) genes that, in turn, bind to phosphorylated JAKs and their receptors to inhibit this pathway, thus preventing over-activation of cytokine-stimulated cells. Therefore, SOCS are a part of a negative feedback loop in the JAK/STAT pathway. JAK stimulation of STATs activates cell proliferation, migration, differentiation, and apoptosis, and deregulation of the inhibitors leads to human diseases, including cancer. Inactivation of the JAK-binding proteins SOCS1 and SSI-1 and activation of the JAK/SKAT pathway have been reported in HCC132,133.\n\nThe ubiquitin proteasome system is involved in cellular protein degradation. After being tagged with ubiquitin, cellular proteins are degraded by the proteasome. The ubiquitin-activating enzyme E1 mediates ATP-dependent transfer of ubiquitin to a ubiquitin-conjugating enzyme (E2), which in turn transfers the ubiquitin either directly to the substrate protein or to a downstream ubiquitin ligase E3, which then ubiquitinates the specific substrate or substrates109. Several cancer-related proteins such as the tumor suppressors p53, p27, pRb, PTEN, the EGF receptor tyrosine kinase, TGFβ, and other cell cycle regulators and oncogenic molecules are regulated by the ubiquitin-proteasome system134. E3 ligases that have been shown to function as tumor suppressors in HCC include the mouse double minute 2 (MDM2) and BRCA1, which target p53 for ubiquitination and degradation135; Smad ubiquitinization regulatory factor-2 (Smurf-2), which targets Smad proteins and the TGFβ receptor complex for degradation136; and the FRA6E fragile site protein Parkin, which targets cyclin E and P38137.\n\nThe liver faces a constant stream of exogenous antigens from the gut reaching it via the portal vein and hence has a unique tolerogenic immune environment. HCC tumor cells developing in this background are able to evade immune surveillance by several mechanisms. Regulatory T cells (Tregs) are a subset of CD4+ T cells that suppress effector CD8 T cells, thus playing an important negative role in anti-tumor immunity138. Several studies have shown that HCC tumor tissues appear to be infiltrated with Tregs and also that patients with HCC have an increased number of circulating Tregs, thus implying that they likely play a pathogenic role in HCC139. Another mechanism of immune suppression in the tumor microenvironment is an increase in immunosuppressive cytokines—such as interleukin-4 (IL-4), IL-5, IL-8, and IL-10—with simultaneous suppression of immune activating cytokines: IL-1, TNF, and interferon gamma140. This unique cytokine signature has been shown to promote tumor metastases, and circulating levels of IL-10 were reported to be associated with poor prognosis140–142. An alternate mechanism of immune evasion that has gained recent attention is modulation of tumor immunity by the programmed cell death-1 (PD-L1/PD-1) immune checkpoint pathway143,144. Increased expression of PD-L1 in tumor cells induces apoptosis of effector T cells and contributes to immune evasion144. Immune checkpoint inhibitors are being increasingly recognized as effective therapeutic option for cancers like melanoma, and a recent early report from a phase I/II study has suggested that, in patients with advanced HCC, the anti-PD-1 monoclonal antibody nivolumab has clinical efficacy without significant toxicity145.\n\nThe origin of self-renewing cells in HCCs is not clearly understood, and, recently, growing evidence supports the novel notion that tumor initiation is likely driven by a subset of cells with stem cell features. These cancer stem cells (CSCs) are considered to be responsible not just for tumor initiation but also for tumor persistence, relapse, and metastasis, thus leading to a more aggressive tumor phenotype146. CSCs also render a tumor chemoresistant and radioresistant, which may explain why HCCs are generally resistant to conventional chemotherapies and also why newer-generation therapies like sorafenib, which do not target CSCs, are associated with frequent tumor relapse after therapy. Hence, identification and characterization of signaling pathways and biomarkers associated with CSCs are priorities for developing new paradigms of molecular cancer therapeutics in the treatment of HCC147. CD133 antigen is considered to be a marker for CSCs, and HCCs with high CD133 expression were associated with poor survival and high recurrence rate148. One of the mechanisms identified in the induction of stemness in HCCs is IL-6-mediated activation of STAT3, which in turn leads to transcriptional activation of CD133149. Another study described CD24 to be a functional marker of liver tumor-initiating cells that drives tumorigenesis through STAT3-mediated regulation of a self-renewing gene NANOG150. Further understanding of the role of CSCs in the pathogenesis of HCC will hopefully unveil new therapeutic targets.\n\n\nConclusions and future directions\n\nHCC is a heterogeneous malignancy resulting from diverse causes of chronic liver injury, with viral hepatitis being the most common etiology. Regardless of the etiology, there appears to be a final common pathway in the pathogenesis of HCC in which repeated hepatocyte damage sets up a vicious cycle of cell death and regeneration which eventually results in genomic instability and initiation of HCC (Figure 2). Recent advances in next-generation sequencing are playing a pivotal role in providing a more comprehensive understanding of the genomic landscape of HCC and in identifying driver mutations (Table 1). Also, recognition of specific molecular pathways commonly involved in HCC initiation and progression is facilitating recognition of novel drug targets for HCC. Currently, sorafenib is the only systemic therapy approved for the management of advanced HCC. A deeper understanding of the molecular pathogenesis of HCC will be instrumental for new drug discovery, which is desperately needed for the thousands of patients with this lethal malignancy.\n\nChronic exposure of the liver to injury from viral hepatitis, alcohol abuse or NASH causes repeated hepatocyte damage and sets up a vicious cycle of cell death and regeneration which eventually results in cirrhosis. The resultant genomic instability leads to initiation of HCC. Step wise accumulation of multiple genetic events including gene rearrangements, somatic mutations, copy number alterations, epigenetic changes and growth factor pathway alterations eventually lead to tumor progression and metastases. Abbreviations: Hep B, hepatitis B; Hep C, hepatitis C; HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis.\n\nThere are several challenges to applying the knowledge gained from understanding the molecular pathogenesis of HCC in the care of patients diagnosed with this malignancy. The significant epidemiologic and molecular heterogeneity of HCC has to be overcome before individualized recommendations can be derived from broad generalizations. With further discovery of molecular subclasses, we can hopefully identify more homogenous subgroups that can be specifically targeted for drug development. Also, there is an urgent need to identify and validate biomarkers that can be used for early, non-invasive diagnosis and for prognostication. The most critical need of the hour is recognition of druggable molecular targets that can promote drug discovery efforts. Further endeavors to create better experimental models, such as patient-derived xenografts, will encourage personalized drug discovery.",
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PubMed Abstract | Publisher Full Text\n\nRoberts LR, Gores GJ: Hepatocellular carcinoma: molecular pathways and new therapeutic targets. Semin Liver Dis. 2005; 25(2): 212–225. PubMed Abstract | Publisher Full Text\n\nBhat M, Sonenberg N, Gores GJ: The mTOR pathway in hepatic malignancies. Hepatology. 2013; 58(2): 810–818. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu L, Cao Y, Chen C, et al.: Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res. 2006; 66(24): 11851–11858. PubMed Abstract | Publisher Full Text\n\nTanabe KK, Lemoine A, Finkelstein DM, et al.: Epidermal growth factor gene functional polymorphism and the risk of hepatocellular carcinoma in patients with cirrhosis. JAMA. 2008; 299(1): 53–60. PubMed Abstract | Publisher Full Text\n\nKaposi-Novak P, Lee JS, Gòmez-Quiroz L, et al.: Met-regulated expression signature defines a subset of human hepatocellular carcinomas with poor prognosis and aggressive phenotype. J Clin Invest. 2006; 116(6): 1582–1595. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLlovet JM, Peña CE, Lathia CD, et al.: Plasma biomarkers as predictors of outcome in patients with advanced hepatocellular carcinoma. Clin Cancer Res. 2012; 18(8): 2290–2300. PubMed Abstract | Publisher Full Text\n\nFrench DM, Lin BC, Wang M, et al.: Targeting FGFR4 inhibits hepatocellular carcinoma in preclinical mouse models. PLoS One. 2012; 7(5): e36713. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDesnoyers LR, Pai R, Ferrando RE, et al.: Targeting FGF19 inhibits tumor growth in colon cancer xenograft and FGF19 transgenic hepatocellular carcinoma models. Oncogene. 2008; 27(1): 85–97. PubMed Abstract | Publisher Full Text\n\nHagel M, Miduturu C, Sheets M, et al.: First Selective Small Molecule Inhibitor of FGFR4 for the Treatment of Hepatocellular Carcinomas with an Activated FGFR4 Signaling Pathway. Cancer Discov. 2015; 5(4): 424–437. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDhanasekaran R, Nakamura I, Hu C, et al.: Activation of the transforming growth factor-β/SMAD transcriptional pathway underlies a novel tumor-promoting role of sulfatase 1 in hepatocellular carcinoma. Hepatology. 2015; 61(4): 1269–1283. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLai JP, Oseini AM, Moser CD, et al.: The oncogenic effect of sulfatase 2 in human hepatocellular carcinoma is mediated in part by glypican 3-dependent Wnt activation. Hepatology. 2010; 52(5): 1680–1689. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLai JP, Sandhu DS, Yu C, et al.: Sulfatase 2 up-regulates glypican 3, promotes fibroblast growth factor signaling, and decreases survival in hepatocellular carcinoma. Hepatology. 2008; 47(4): 1211–1222. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYoshiji H, Kuriyama S, Noguchi R, et al.: Angiopoietin 2 displays a vascular endothelial growth factor dependent synergistic effect in hepatocellular carcinoma development in mice. Gut. 2005; 54(12): 1768–1775. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPark YN, Kim YB, Yang KM, et al.: Increased expression of vascular endothelial growth factor and angiogenesis in the early stage of multistep hepatocarcinogenesis. Arch Pathol Lab Med. 2000; 124(7): 1061–1065. PubMed Abstract\n\nMuto J, Shirabe K, Sugimachi K, et al.: Review of angiogenesis in hepatocellular carcinoma. Hepatol Res. 2015; 45(1): 1–9. PubMed Abstract | Publisher Full Text\n\nKim KW, Bae SK, Lee OH, et al.: Insulin-like growth factor II induced by hypoxia may contribute to angiogenesis of human hepatocellular carcinoma. Cancer Res. 1998; 58(2): 348–351. PubMed Abstract\n\nCannito S, Turato C, Paternostro C, et al.: Hypoxia up-regulates SERPINB3 through HIF-2α in human liver cancer cells. Oncotarget. 2015; 6(4): 2206–2221. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLuo D, Wang Z, Wu J, et al.: The role of hypoxia inducible factor-1 in hepatocellular carcinoma. Biomed Res Int. 2014; 2014: 409272. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoberts AB, Wakefield LM: The two faces of transforming growth factor beta in carcinogenesis. Proc Natl Acad Sci U S A. 2003; 100(15): 8621–8623. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCoulouarn C, Factor VM, Thorgeirsson SS: Transforming growth factor-beta gene expression signature in mouse hepatocytes predicts clinical outcome in human cancer. Hepatology. 2008; 47(6): 2059–2067. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGiannelli G, Bergamini C, Fransvea E, et al.: Laminin-5 with transforming growth factor-beta1 induces epithelial to mesenchymal transition in hepatocellular carcinoma. Gastroenterology. 2005; 129(5): 1375–1383. PubMed Abstract | Publisher Full Text\n\nGiannelli G, Villa E, Lahn M: Transforming growth factor-β as a therapeutic target in hepatocellular carcinoma. Cancer Res. 2014; 74(7): 1890–1894. PubMed Abstract | Publisher Full Text\n\nZheng X, Gai X, Han S, et al.: The human sulfatase 2 inhibitor 2,4-disulfonylphenyl-tert-butylnitrone (OKN-007) has an antitumor effect in hepatocellular carcinoma mediated via suppression of TGFB1/SMAD2 and Hedgehog/GLI1 signaling. Genes Chromosomes Cancer. 2013; 52(3): 225–236. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCalvisi DF, Ladu S, Gorden A, et al.: Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology. 2006; 130(4): 1117–1128. PubMed Abstract | Publisher Full Text\n\nYasuda E, Kumada T, Takai S, et al.: Attenuated phosphorylation of heat shock protein 27 correlates with tumor progression in patients with hepatocellular carcinoma. Biochem Biophys Res Commun. 2005; 337(1): 337–342. PubMed Abstract | Publisher Full Text\n\nBurger AM, Seth AK: The ubiquitin-mediated protein degradation pathway in cancer: therapeutic implications. Eur J Cancer. 2004; 40(15): 2217–2229. PubMed Abstract | Publisher Full Text\n\nYang Y, Li CC, Weissman AM: Regulating the p53 system through ubiquitination. Oncogene. 2004; 23(11): 2096–2106. PubMed Abstract | Publisher Full Text\n\nZhang Y, Chang C, Gehling DJ, et al.: Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase. Proc Natl Acad Sci U S A. 2001; 98(3): 974–979. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang F, Denison S, Lai JP, et al.: Parkin gene alterations in hepatocellular carcinoma. Genes Chromosomes Cancer. 2004; 40(2): 85–96. PubMed Abstract | Publisher Full Text\n\nYu P, Fu YX: Tumor-infiltrating T lymphocytes: friends or foes? Lab Invest. 2006; 86(3): 231–245. PubMed Abstract | Publisher Full Text\n\nZhao HQ, Li WM, Lu ZQ, et al.: Roles of Tregs in development of hepatocellular carcinoma: a meta-analysis. World J Gastroenterol. 2014; 20(24): 7971–7978. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBudhu A, Forgues M, Ye QH, et al.: Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment. Cancer Cell. 2006; 10(2): 99–111. PubMed Abstract | Publisher Full Text\n\nHattori E, Okumoto K, Adachi T, et al.: Possible contribution of circulating interleukin-10 (IL-10) to anti-tumor immunity and prognosis in patients with unresectable hepatocellular carcinoma. Hepatol Res. 2003; 27(4): 309–314. PubMed Abstract | Publisher Full Text\n\nChan SL, Mo FK, Wong CS, et al.: A study of circulating interleukin 10 in prognostication of unresectable hepatocellular carcinoma. Cancer. 2012; 118(16): 3984–3992. PubMed Abstract | Publisher Full Text\n\nWu K, Kryczek I, Chen L, et al.: Kupffer cell suppression of CD8+ T cells in human hepatocellular carcinoma is mediated by B7-H1/programmed death-1 interactions. Cancer Res. 2009; 69(20): 8067–8075. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShi F, Shi M, Zeng Z, et al.: PD-1 and PD-L1 upregulation promotes CD8+ T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients. Int J Cancer. 2011; 128(4): 887–896. PubMed Abstract | Publisher Full Text\n\nEl-Khoueiry AB, Melero I, Crocenzi TS, et al.: Phase I/II safety and antitumor activity of nivolumab in patients with advanced hepatocellular carcinoma (HCC): CA209-040. J Clin Oncol. 2015; 33(18): (suppl, abstr LBA101). Reference Source\n\nYamashita T, Wang XW: Cancer stem cells in the development of liver cancer. J Clin Invest. 2013; 123(5): 1911–1918. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarquardt JU, Factor VM, Thorgeirsson SS: Epigenetic regulation of cancer stem cells in liver cancer: current concepts and clinical implications. J Hepatol. 2010; 53(3): 568–577. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSong W, Li H, Tao K, et al.: Expression and clinical significance of the stem cell marker CD133 in hepatocellular carcinoma. Int J Clin Pract. 2008; 62(8): 1212–1218. PubMed Abstract | Publisher Full Text\n\nWon C, Kim BH, Yi EH, et al.: Signal transducer and activator of transcription 3-mediated CD133 up-regulation contributes to promotion of hepatocellular carcinoma. Hepatology. 2015; 62(4): 1160–1173. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLee TK, Castilho A, Cheung VC, et al.: CD24+ liver tumor-initiating cells drive self-renewal and tumor initiation through STAT3-mediated NANOG regulation. Cell Stem Cell. 2011; 9(1): 50–63. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13814",
"date": "12 May 2016",
"name": "Yun-Han Lee",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13815",
"date": "12 May 2016",
"name": "Xin Wei 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",
"responses": []
},
{
"id": "13816",
"date": "12 May 2016",
"name": "Irene Oi-Lin Ng",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13817",
"date": "12 May 2016",
"name": "Stephen L Chan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-879
|
https://f1000research.com/articles/5-877/v1
|
12 May 16
|
{
"type": "Review",
"title": "Cell cycle regulated transcription: from yeast to cancer",
"authors": [
"Christopher J. McInerny"
],
"abstract": "Recent studies have revealed exciting new functions for forkhead transcription factors in cell proliferation and development. Cell proliferation is a fundamental process controlled by multiple overlapping mechanisms, and the control of gene expression plays a major role in the orderly and timely division of cells. This occurs through transcription factors regulating the expression of groups of genes at particular phases of the cell division cycle. In this way, the encoded gene products are present when they are required. This review outlines recent advances in our understanding of this process in yeast model systems and describes how this knowledge has informed analysis in more developmentally complex eukaryotes, particularly where it is relevant to human disease.",
"keywords": [
"Cell cycle",
"yeast",
"transcription",
"Cell proliferation",
"cancer"
],
"content": "Introduction\n\nThe accurate duplication of a cell is essential for successful cell proliferation. Many cellular mechanisms control the cell division cycle, and some operate in parallel and show redundancy. This “belt and braces” approach contributes to the incredible fidelity of the process. The control mechanisms range from changes in protein activity (for example, through modifications in phosphorylation status) to changes in protein stability or localisation.\n\nAnother major level of control is through the regulation of gene transcription, where genes or groups of genes are transcribed at particular cell cycle times in order to ensure that protein products are produced when they are required. In some cases, the protein products have structural or enzymatic functions specific to a certain cell cycle phase. In other cases, they have regulatory roles such as activating other proteins important for cell division or controlling the transcription of genes required later in the cell cycle. There are also a few examples of protein products whose presence at the “wrong” cell cycle time is deleterious to the cell.\n\n\nYeasts\n\nRegulation of gene transcription through the cell division cycle has been studied most extensively in two yeast species: the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe1–3 (Figure 1). In both yeasts, groups of genes are transcribed at different cell cycle phases. The expression of each group of genes is controlled by a different transcription factor. Each transcription factor binds to DNA enhancer sequences that are present only in the promoter regions of genes transcribed at that cell cycle phase, ensuring cell cycle-specific gene transcription.\n\nIn each yeast species, the principal transcription factors are shown, and the downstream transcription factor(s) that they regulate, either positively or negatively, are indicated by arrows. Where similar transcription factors are present in the two species, they are shown in the same colour. In budding yeast, a reasonably complete network exists, whereby consecutive regulation of transcription factors encompasses the whole cell cycle. In contrast, the network in fission yeast is more limited.\n\nIn budding yeast, where the most comprehensive transcription network has been elucidated and described, there exists a series of transcription factors that stimulate or repress the expression of consecutive groups of genes1 (Figure 1). In some cases, the genes encode transcription factors that stimulate or repress the expression of the next group of gene transcription in the cell cycle. Thus, the groups of gene expression can be functionally linked, and this linkage encompasses the whole cell cycle. In fission yeast, a more limited network has been described, and three groups of gene expression are observed (Figure 1); in only one case is there evidence that one group of gene expression controls the expression of the next group2,3.\n\nStrikingly, however, a number of these transcription factors are evolutionarily conserved with related proteins found in the two yeast species. Furthermore, these same transcription factors have been identified in more developmentally complex eukaryotes (including humans) where they too control cell cycle gene expression1–3. Examples include (1) the forkhead transcription factors which control expression of two groups of genes in the cell cycle, one during the mitotic (M) phase and the other at the end of the M phase and the beginning of the Gap 1 (G1) period, and (2) the MBF-Nrm1 transcription factor complex which regulates gene expression at the end of the G1 period and the start of the synthesis (S) phase. The remainder of this review will focus on these two systems, first by describing them in more detail in each model yeast and then by explaining their function in human cells and in disease.\n\n\nForkhead transcription factor gene regulation\n\nThe role of forkhead transcription factors in cell cycle-regulated gene expression was first described in budding yeast. The forkhead transcription factor Fkh2, in combination with the MADS box protein Mcm1, controls gene expression during M phase1–5 (Figure 1). In addition to regulating the expression of M phase genes, Fkh2-Mcm1 controls the activity of the transcription factors Ace2 and Swi5, which regulate gene expression later in the cell cycle during G1.\n\nSimilar mechanisms have been identified in fission yeast, where the forkhead transcription factor Fkh2, in combination with the MADS box protein Mbx1, regulates M-G1 gene expression as well as the subsequent group of gene expression in G1 through the regulation of the Ace2 transcription factor6,7 (Figure 1).\n\nSuch conservation of control mechanisms between two distantly related yeasts suggests that forkhead transcription factors are important. Consistent with this suggestion, similar control processes have been identified in more developmentally complex eukaryotes. For example, the human forkhead transcription factors FOXO1 and FOXO2 regulate cell cycle gene transcription that is critical for proliferation8,9. Furthermore, the medical importance of FOXO1 and FOXO2 has been established, as these transcription factors have roles in a wide range of human medical conditions, including cancer, obesity, diabetes, autoimmune disease, and ageing10–14. Recently, the formation of blood vessels through the control of the metabolism and proliferation of vascular endothelial cells has been shown to be under the control of FOXO115. Indeed, forkhead transcription factors are now seen as promising therapeutic targets for a wide variety of conditions16,17.\n\n\nMBF-Nrm1 gene regulation\n\nAs one of the first cell cycle transcription factors to be identified in yeasts, the MBF complex has a long history1–3. In budding yeast, it is composed of Swi4 and Mbp1 and controls the expression of a large group of genes in late G1 and early S phase (Figure 1). MBF in fission yeast is composed of related proteins (including Cdc10, Res1, and Res2) and similarly controls gene expression at G1/S. MBF in both yeasts is controlled by Nrm118–21.\n\nThe human functional equivalent to MBF is E2F, which, along with pocket proteins such as the retinoblastoma protein Rb, controls the expression of genes at G1/S9,22. The expression of these genes is deregulated in many types of cancer, as they allow cancer cells to divide in the absence of growth factors, thereby making them insensitive to signals that normally inhibit growth23,24. E2F family members are now potential therapeutic targets for certain malignancies25.\n\nRecent experiments have revealed further mechanisms by which deregulated gene transcription can contribute to abnormal cell cycles, the hallmark of cancer and tumorigenesis. Caetano et al.26 have shown that in fission yeast deregulation of G1/S gene expression increases DNA replication errors. This demonstrates that faulty transcription of certain genes under the control of MBF can have a profound impact on genome stability. It seems likely that similar mechanisms operate in human cells and that these may be possible targets for therapeutics.\n\n\nFuture challenges\n\nThe examples described here show how yeast model systems have been used to identify proteins that control the cell division cycle in all eukaryotes, including humans. That these proteins have subsequently been shown to be useful therapeutic targets suggests that this avenue of study will continue to be useful in the future. It seems likely that more transcription factors with cell cycle-specific functions remain to be identified and, moreover, that yeasts will continue to offer an excellent system in which to discover and characterise them.",
"appendix": "Competing interests\n\n\n\nThe author declares that he has no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHaase SB, Wittenberg C: Topology and control of the cell-cycle-regulated transcriptional circuitry. Genetics. 2014; 196(1): 65–90. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBähler J: Cell-cycle control of gene expression in budding and fission yeast. Annu Rev Genet. 2005; 39: 69–94. PubMed Abstract | Publisher Full Text\n\nMcInerny CJ: Cell cycle regulated gene expression in yeasts. Adv Genet. 2011; 73: 51–85. PubMed Abstract | Publisher Full Text\n\nBoros J, Lim FL, Darieva Z, et al.: Molecular determinants of the cell-cycle regulated Mcm1p-Fkh2p transcription factor complex. Nucleic Acids Res. 2003; 31(9): 2279–88. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDarieva Z, Clancy A, Bulmer R, et al.: A competitive transcription factor binding mechanism determines the timing of late cell cycle-dependent gene expression. Mol Cell. 2010; 38(1): 29–40. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPapadopoulou K, Ng SS, Ohkura H, et al.: Regulation of gene expression during M-G1-phase in fission yeast through Plo1p and forkhead transcription factors. J Cell Sci. 2008; 121(Pt 1): 38–47. PubMed Abstract | Publisher Full Text\n\nSuárez MB, Alonso-Nuñez ML, del Rey F, et al.: Regulation of Ace2-dependent genes requires components of the PBF complex in Schizosaccharomyces pombe. Cell Cycle. 2015; 14(19): 3124–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMurakami H, Aiba H, Nakanishi M, et al.: Regulation of yeast forkhead transcription factors and FoxM1 by cyclin-dependent and polo-like kinases. Cell Cycle. 2010; 9(16): 3233–42. PubMed Abstract | Publisher Full Text\n\nGrant GD, Brooks L 3rd, Zhang X, et al.: Identification of cell cycle-regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors. Mol Biol Cell. 2013; 24(23): 3634–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDansen TB, Kalkhoven E: Targeting FOXO1 as an option to treat obesity? Cell Cycle. 2015; 14(16): 2558. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartins R, Lithgow GJ, Link W: Long live FOXO: unraveling the role of FOXO proteins in aging and longevity. Aging Cell. 2016; 15(2): 196–207. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCoomans de Brachène A, Demoulin JB: FOXO transcription factors in cancer development and therapy. Cell Mol Life Sci. 2016; 73(6): 1159–72. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nZhang T, Kim DH, Xiao X, et al.: FoxO1 Plays an Important Role in Regulating β-Cell Compensation for Insulin Resistance in Male Mice. Endocrinology. 2016; 157(3): 1055–70. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTosiek MJ, Fiette L, El Daker S, et al.: IL-15-dependent balance between Foxp3 and RORγt expression impacts inflammatory bowel disease. Nat Commun. 2016; 7: 10888. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWilhelm K, Happel K, Eelen G, et al.: FOXO1 couples metabolic activity and growth state in the vascular endothelium. Nature. 2016; 529(7585): 216–20. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHill R, Kalathur RK, Callejas S, et al.: A novel phosphatidylinositol 3-kinase (PI3K) inhibitor directs a potent FOXO-dependent, p53-independent cell cycle arrest phenotype characterized by the differential induction of a subset of FOXO-regulated genes. Breast Cancer Res. 2014; 16(6): 482. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBullock M: FOXO factors and breast cancer: outfoxing endocrine resistance. Endocr Relat Cancer. 2016; 23(2): R113–30. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nde Bruin RA, Kalashnikova TI, Chahwan C, et al.: Constraining G1-specific transcription to late G1 phase: the MBF-associated corepressor Nrm1 acts via negative feedback. Mol Cell. 2006; 23(4): 483–96. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nOstapenko D, Solomon MJ: Anaphase promoting complex-dependent degradation of transcriptional repressors Nrm1 and Yhp1 in Saccharomyces cerevisiae. Mol Biol Cell. 2011; 22(13): 2175–84. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nOfir A, Hofmann K, Weindling E, et al.: Role of a Candida albicans Nrm1/Whi5 homologue in cell cycle gene expression and DNA replication stress response. Mol Microbiol. 2012; 84(4): 778–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTravesa A, Kalashnikova TI, de Bruin RA, et al.: Repression of G1/S transcription is mediated via interaction of the GTB motifs of Nrm1 and Whi5 with Swi6. Mol Cell Biol. 2013; 33(8): 1476–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPolager S, Ginsberg D: E2F - at the crossroads of life and death. Trends Cell Biol. 2008; 18(11): 528–35. PubMed Abstract | Publisher Full Text\n\nChen HZ, Tsai SY, Leone G: Emerging roles of E2Fs in cancer: an exit from cell cycle control. Nat Rev Cancer. 2009; 9(11): 785–97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEngelmann D, Pützer BM: The dark side of E2F1: in transit beyond apoptosis. Cancer Res. 2012; 72(3): 571–5. PubMed Abstract | Publisher Full Text\n\nPark SA, Platt J, Lee JW, et al.: E2F8 as a novel therapeutic target for lung cancer. J Natl Cancer Inst. 2015; 107(9): pii: djv151. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCaetano C, Limbo O, Farmer S, et al.: Tolerance of deregulated G1/S transcription depends on critical G1/S regulon genes to prevent catastrophic genome instability. Cell Rep. 2014; 9(6): 2279–89. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "13804",
"date": "12 May 2016",
"name": "Jonathan Millar",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13805",
"date": "12 May 2016",
"name": "Jim Karagiannis",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-877
|
https://f1000research.com/articles/5-876/v1
|
12 May 16
|
{
"type": "Review",
"title": "Intracerebral haemorrhage in Down syndrome: protected or predisposed?",
"authors": [
"Lewis Buss",
"Elizabeth Fisher",
"John Hardy",
"Dean Nizetic",
"Jurgen Groet",
"Laura Pulford",
"André Strydom",
"Lewis Buss",
"Elizabeth Fisher",
"John Hardy",
"Dean Nizetic",
"Jurgen Groet",
"Laura Pulford"
],
"abstract": "Down syndrome (DS), which arises from trisomy of chromosome 21, is associated with deposition of large amounts of amyloid within the central nervous system. Amyloid accumulates in two compartments: as plaques within the brain parenchyma and in vessel walls of the cerebral microvasculature. The parenchymal plaque amyloid is thought to result in an early onsetAlzheimer’s disease (AD) dementia, a phenomenon so common amongst people with DS that it could be considered a defining feature of the condition. The amyloid precursor protein (APP) gene lies on chromosome 21 and its presence in three copies in DS is thought to largely drive the early onset AD. In contrast, intracerebral haemorrhage (ICH), the main clinical consequence of vascular amyloidosis, is a more poorly defined feature of DS. We review recent epidemiological data on stroke (including haemorrhagic stroke) in order to make comparisons with a rare form of familial AD due to duplication (i.e. having three copies) of the APP region on chromosome 21, here called ‘dup-APP’, which is associated with more frequent and severe ICH. We conclude that although people with DS are at increased risk of ICH, this is less common than in dup-APP, suggesting the presence of mechanisms that act protectively. We review these mechanisms and consider comparative research into DS and dup-APP that may yield further pathophysiological insight.",
"keywords": [
"Intracerebral haemorrhage",
"Down syndrome",
"trisomy",
"Alzheimer’s disease",
"Cerebral amyloid angiopathy"
],
"content": "Introduction\n\nDown syndrome (DS), which is due to an extra copy of chromosome 21, is strongly associated with early onset Alzheimer’s disease (AD)1. This is likely due to the presence of three copies of the gene coding for the amyloid precursor protein (APP) situated on chromosome 21, resulting in typical pathological features of AD, including senile plaques, composed of small, insoluble fragments of APP referred to as amyloid β (Aβ), formed after cleavage by specific secretase enzymes. The ensuing neurocognitive decline is a striking clinical feature of DS.\n\nCerebral amyloid angiopathy (CAA) results from the deposition of amyloid within the walls of leptomeningeal and cerebral blood vessels2 and is present in more than 80% of AD brains at post mortem3. As in AD, this amyloid derives from APP and is also composed of Aβ fragments. The process of vascular amyloid deposition is largely silent; however, when severe, it may set off a cascade of events resulting in intracerebral haemorrhage (ICH), the main clinical consequence of CAA. CAA-related haemorrhages tend to affect the elderly and occur multiply and in cortical and subcortical (lobar) regions4. Like AD, CAA occurs frequently in DS5; however, unlike AD dementia, CAA-ICH is not a well-established clinical phenomenon in people with DS.\n\nIf CAA-ICH is over-represented in people with DS compared with the euploid population, it is important as an avenue for research and also to clinicians to provide more appropriate care to this group. In this review, we consider the rates of CAA and ICH in individuals with DS and compare these with sporadic AD as well as a specific form of familial AD due to duplication of the APP region on chromosome 21. Finally, we consider potential mechanisms for apparent differences between these groups.\n\n\nIntracerebral haemorrhage in Down syndrome: epidemiology\n\nUntil recently, data on CAA-ICH in DS have been limited. There are seven case reports of people with DS suffering severe ICH6–11. Their paucity and noteworthiness suggest that ICH is not part of the experience of clinicians caring for people with DS. However, several mortality studies have reported increased incidence of cerebrovascular events in people with DS12,13 but failed to distinguish between ischaemic and haemorrhagic stroke types.\n\nMore recently, Sobey et al. reported population-level data on cardiovascular events in 4081 people with DS and 16,324 age-matched controls14. Both ischaemic (risk ratio [RR] = 3.76, 95% confidence interval [CI] 2.39, 5.92) and haemorrhagic (RR = 3.31, 95% CI 1.95, 5.60) strokes are reported as more common amongst people with DS than non-DS controls. The incidence rates for ‘any stroke’ were 1.3% in males and 2.3% in women aged 19 to 50. For those over 51 years, the corresponding values were 11.3% in males and 8.2% in women. For haemorrhagic stroke, the values were 3.8% in males and 3.3% in women older than 51. However, when corrected for existing cardiovascular risk factors (including hypertension, diabetes, smoking, cardiac arrhythmia, sleep apnoea, congenital heart disease, pulmonary hypertension, and Moyamoya disease), the increased risk is largely attenuated for ischaemic but not for haemorrhagic stroke. The authors propose that this excess risk may be accounted for by factors not adjusted for in the regression analysis, such as misclassification of ischaemic-haemorrhagic transformation or anticoagulation, but it is also possible that some of this increased risk for haemorrhagic stroke may be explained by the deposition of vascular amyloid seen in DS, supporting the view that CAA-ICH is relatively common in DS.\n\n\nAmyloid precursor protein duplication\n\nThe genetic abnormality in DS thought to underpin AD and CAA is the triplication of the APP gene, along with the rest of chromosome 21. It is proposed that this increased ‘dose’ of APP provides extra substrate for Aβ production, which then is deposited as senile plaques in AD or vascular amyloid in CAA.\n\nAPP is cleaved to Aβ fragments by γ-secretase enzymes whose catalytic subunit is coded for by PSEN1 and PSEN2 genes. There are many well-documented familial forms of AD caused by missense mutations in PSEN1, PSEN2, and the APP gene15 that modulate APP processing and increase Aβ deposition. Similarly, hereditary forms of CAA, such as the Dutch type CAA, result from missense mutations in the same three genes16.\n\nIn addition, a novel genetic form of AD has been recognised in the last 10 years arising from small internal chromosome 21 duplications17–25. These rare copy number variants all result in three copies of APP, collectively known as duplication of APP (dup-APP)26, and lead to an APP overdose. In this sense, dup-APP differs from other forms of familial AD that are the result of point mutations in APP, PSEN1, or PSEN2. Meaningful comparison can be made with DS, as an additional copy of APP is present in both diseases; DS differs from dup-APP only in the number of other genes on chromosome 21 that are also trisomic.\n\nThe phenotype of dup-APP is one of a highly penetrant AD dementia (frequently associated with seizures17,18,22) with an onset age of between 39 and 64 years26. Significantly, carriers of dup-APP suffer a strikingly high rate of ICH. We estimate that this occurs in approximately a third of the published cases (Table 1 and Figure 1). These haemorrhages are typical of CAA-ICH: multiple and in a lobar distribution. They represent a serious clinical event and are a frequent cause of death in those affected.\n\nThe third column gives the most conservative estimate of the proportion of cases, known to harbour duplication (i.e. having three copies) of the amyloid precursor protein region on chromosome 21 (dup-APP), that are also affected by intracerebral haemorrhage (ICH).\n\nPhenotype with respect to dementia (black crescent) and intracerebral haemorrhage (ICH) (red crescent) is shown. Genotype is reported as APP duplication present (dup), absent (-), or not determined (ND). The French kindreds (Wallon et al.24) are not shown, as there are insufficient data provided for this purpose. Owing to limitations of the original article, it was not possible to report the genotypes for the Finnish kindred: nine of the affected individuals carry dup-APP, but it is not reported which of the family members this applies to.\n\nComparison of DS and dup-APP groups shows that the additional copy of the APP gene is sufficient to produce both early onset AD and CAA-ICH. However, although haemorrhagic stroke appears to occur in a significant proportion of elderly people with DS (3.3% to 3.8%)14, people with dup-APP are much more profoundly affected; the occurrence rate is approximately 30% (i.e. nearly 10 times higher than in DS). This suggests that triplication of the rest of chromosome 21 may provide partial protection against the pro-haemorrhagic effects of APP duplication.\n\n\nPathophysiological insights\n\nAβ is primarily deposited in the adventitia and media of involved arterioles, and severity of CAA is classified according to spread through the vessel wall: mild CAA is defined as Aβ in the adventitia and some deposits between smooth muscle cells in the media, which are restricted to the tunica media without death of smooth muscle cells. Moderate CAA involves replacement of smooth muscle cells by Aβ and thickening of the media without disruption of the blood-brain barrier (BBB). Severe CAA is defined as extensive Aβ deposition with fragmentation or double-barrelling of the vessel wall, fibrinoid necrosis, and formation of aneurysms27.\n\nThe order in which vessels are affected typically follows a particular sequence; the leptomeningeal arteries are the first to show signs of pathology, followed by penetrating arterioles in the neocortical grey matter. Furthermore, vessels in the posterior regions of the brain (such as the occipital lobe) are especially affected, although the frontal cortex has also been named as a relatively early site, followed by vessels of the olfactory cortex, hippocampus, and cerebellum, while deep grey and white matter are usually spared3,28,29.\n\nICH due to CAA is typically lobar, and recurrent or multiple, and may occur in the absence of other risk factors for haemorrhage, such as hypertension. Once haemorrhage has occurred, the result is extensive neuronal death as well as a local immune response from microglia, astrocytes, and other immune cells. The salience of ICH in the phenotype of dup-APP is mirrored by the severity of underlying CAA reported in neuropathology studies. In all 13 cases of dup-APP in which neuropathology has been studied (age range of 48 to 58 years), the histological grade of CAA was moderate to severe and CAA was found in every brain reported17,18,20,23. By contrast, CAA is not a universal finding in people with DS (Figure 2, data taken from 5). Although most post mortem examinations on people with DS over the age of 50 show CAA from a moderate to severe degree5,30–32, a significant proportion (approximately one in five) (Figure 1) remains completely unaffected by CAA.\n\nAge ranges are indicated above charts. Data are reproduced from 5.\n\nThese observations suggest a complex relationship between increased APP gene dosage and CAA-ICH; individuals with DS show an increased prevalence of CAA-ICH compared with the euploid population but lower prevalence compared with dup-APP individuals. This suggests some degree of protection, but the mechanisms mediating this relationship are as yet unknown. We will consider the possibilities in the following sections.\n\n\nAβ 40 versus Aβ 42\n\nThe 40-amino-acid peptide Aβ (Aβ 40) is more soluble than the longer Aβ 42 peptide. Aβ 40 tends to be the major form of Aβ in the artery walls in CAA, whereas Aβ 42 is more prominent in plaques. The ratio of Aβ 40/42 seems to determine to some extent whether Aβ is deposited in brain parenchyma or in the vessel walls. In mutations where Aβ 42 is the prominent form of Aβ, such as the Indiana and London APP mutations, vascular amyloid seems to be a less prominent feature than parenchymal plaques33. In contrast, a high Aβ 40/42 ratio may promote CAA3, as seen in CAA Dutch type34.\n\nCellular studies using cortical neurones generated from induced pluripotent stem cells harbouring APP duplication or DS (trisomy of chromosome 21) have addressed the issue of Aβ 40/42 ratio. Both dup-APP35,36 and trisomy 2137,38 cells overproduce Aβ peptide compared with control cells, although the relative overproduction has not been compared in the same study protocol. Surprisingly, in dup-APP cells, the Aβ 40/Aβ 42 ratio is unchanged compared with control cells, which is also the case in mouse studies of overexpression of wild-type APP, resulting in an increase of both Aβ 40 and Aβ 42 with the ratio preserved34. In contrast, trisomic cells exhibit an increased Aβ 40/Aβ 42 ratio compared with dup-APP and control cells35.\n\nIn post mortem studies, the composition of vascular and parenchymal amyloid appears very similar in dup-APP and DS brains when stained with antibodies specific to either Aβ 40 or Aβ 42. Older individuals with DS30,31 and dup-APP cases17,19 have extensive Aβ 40 deposition in vascular walls, preceded temporarily by low-level Aβ 42 deposition in DS31. Parenchymal plaques are present in both groups to a similar extent and stain mostly prominently for Aβ 42.\n\nGiven the cellular data, it seems that the Aβ 40/42 ratio may be elevated in DS compared with dup-APP and controls; however, this is not reflected strongly in histopathology studies, which do not suggest significant differences in neuropathology between DS and dup-APP. An increased Aβ 40/42 ratio in DS would predict more severe CAA and predisposition to ICH; however, it does not explain the relative protection compared with dup-APP. Other mechanisms may be at play.\n\n\nAβ clearance in Down syndrome\n\nIncreased amyloid in the brain can be the result of either increased production or reduced clearance. As discussed in the preceding section, there is increased production of amyloid in both dup-APP and DS because of the presence of an extra copy of the APP gene in both cases. The Aβ 40/Aβ 42 ratios do not explain the apparent difference between dup-APP and DS in severity of CAA and prevalence of ICH. It is possible that a difference in clearance of vascular amyloid is the key factor.\n\nAβ is cleared from the brain by several pathways: (1) endocytosis by astrocytes and microglial cells39, (2) enzymatic degradation40, or (3) removal through the BBB41 or along peri-arterial spaces42.\n\nMicroglia are the brain’s tissue macrophages; they have been shown to clear Aβ by endocytosis and internal degradation39. However, their exact role in this process is still poorly understood. Recent post mortem studies of brains from individuals with DS who were not older than 40 (i.e. before onset of AD) showed a heightened neuroinflammatory response, which was further increased in older individuals with DS and AD. Microglial cell activation increases with age in DS43 but may be lower compared with sporadic AD cases, despite higher levels of Aβ accumulation, and DS brains were characterised by a unique inflammatory phenotype associated with the formation of immune complexes (M2b)44. The authors hypothesised that accumulation of CAA may result in vascular leakage, with extravasation of IgG into the brain, which in turn may promote the M2b phenotype. Intriguingly, previous work by this group showed that an M2b inflammatory phenotype induced by IgG infusions into brains of an amyloid mouse model promoted clearance of amyloid deposits, suggesting a protective mechanism45. However, if specific changes in neuroinflammation and microglial cells exist in DS, they have not yet been fully investigated and neither has the nature of these mechanisms been explored in dup-APP. Furthermore, since the bulk of extracellular Aβ clearance is via the BBB or interstitial fluid flow46, it seems unlikely to be the main factor accounting for the relative protection against CAA-ICH in DS as compared with dup-APP.\n\nClearance of Aβ locally is also performed by the cerebrovascular smooth muscle cells and astrocytes through the low-density lipoprotein receptor-related protein-1 (LRP1)-mediated endocytic pathway47. The levels of LRP1 are reduced in patients with AD, and LRP1 levels also decline with age48. Recently, assays were developed to model these processes by using induced pluripotent stem cell models49. The effects of dup-APP and trisomy 21 on these processes are yet to be studied.\n\nPhysiological degradation of Aβ involves metallopeptidases such as neprilysin (NEP)46. NEP degradation of Aβ seems to be protective against CAA50. NEP expression in vascular smooth muscle cells is inversely correlated with degree of vascular Aβ51,52, and a polymorphism in the NEP promotor region that may reduce NEP transcription levels is associated with more severe CAA53. There is little published research on enzymatic degradation of Aβ in DS. However, one study by Miners et al. (2010) showed NEP levels to be increased in DS brains (age range of 10 to 80 years) compared with non-DS controls, and NEP level was strongly correlated with insoluble Aβ concentration54. This contrasts with evidence from sporadic AD showing decreased NEP immunoreactivity compared with age-matched controls55. It is possible that in DS there is greater capacity for NEP expression conferring some protective effect against CAA.\n\nTransport of Aβ across the BBB is receptor mediated. The low-density lipoprotein pathway transports Aβ from the brain interstitial and cerebrospinal fluid compartments into the circulation56. To the best of our knowledge, specific abnormalities of the BBB have not been demonstrated in DS; however, lipid processing is known to be abnormal57, indirectly supporting the idea that BBB-mediated efflux of Aβ from the brain could be altered in this group.\n\nA proportion of neuronally produced Aβ flows with the interstitial fluid along perivascular spaces to be excreted into the cerebrospinal fluid and drained into the systemic circulation42. Although the contribution of perivascular drainage to CAA is by no means clear, it has been proposed to be a compensatory mechanism when other routes fail, and this may underlie the strong association between age and the development of CAA and AD pathology in the general population. Perivascular drainage is proposed to rely on counter-current flow of lymphatics driven by the arterial pulsation58. As the arteries are increasingly affected by atherosclerosis or inflammation during ageing, they become more rigid with less effective contractile function and perivascular drainage. As individuals with DS appear to be somewhat protected against atherosclerosis57, this could be another protective mechanism, resulting in better Aβ drainage and less severe CAA.\n\n\nOther potentially protective mechanisms\n\nDS may be associated with several other protective mechanisms. It is possible that DS differs from dup-APP in the response to Aβ-related cell damage. Evidence from mouse model studies shows that immunotherapy against amyloid increases CAA and may also result in increased micro-haemorrhages59, suggesting an important role for the immune system in the pathophysiology of CAA. The innate immune system may differ in DS, thus affecting response to AB deposition. This possibility remains to be explored. Furthermore, the effect of free radicals may contribute to vascular damage, and although oxidative stress is a prominent feature of DS, it has been shown that increased activity of some anti-oxidant enzymes such as superoxide dismutase (SOD1, encoded on chromosome 21 and triplicated in DS) is associated with less cognitive decline, suggesting another potential protective mechanism60,61. In contrast, experiments using the Tg2576 mouse model have resulted in the suggestion that Aβ-induced oxidative stress causes DNA damage and excess opening of TRPM2 calcium channels, leading to calcium overload, which in turn results in endothelial dysfunction62.\n\nSpecific apolipoprotein E (APOE) genotypes (ε4 and ε2) are known risk factors for more severe CAA and ICH in the general population63–65. Two of the seven cases of ICH in DS report APOE genotype, both carrying high-risk variants (ε2/ε47 and ε4/ε49). This contrasts with dup-APP, where all 11 cases of ICH in which APOE genotype is reported carry a low-risk variant (ε3/ε3 n = 9, ε3/ε4 n = 2). These very limited data suggest an importance of APOE genotype as a risk factor for CAA-ICH in DS and possible APOE independence of ICH in dup-APP; further investigation is needed. However, it is noteworthy that APOE is mapped to chromosome 19, not chromosome 21, and this by itself is unlikely to explain differences between DS and dup-APP groups. In this regard, the ATP binding-cassette G1 (ABCG1) gene may be a more relevant candidate, as it is located on chromosome 21 and is thought to be responsible for cholesterol efflux onto apolipoproteins66. However, cellular studies provide conflicting evidence suggesting that ABCG1 overexpression may increase67 or reduce68 Aβ production. Evidence from mouse models is also conflicting. One study examined transgenic mice with a sixfold overexpression of ABCG1 that did not exhibit increased levels of Aβ69; by contrast, APOE ε4 mice treated with bexarotene, an agent that indirectly upregulates ABCG1 and ABCA1, reversed hippocampal Aβ 42 deposition70. The evidence is unclear and its relation to CAA and ICH even more so.\n\nFinally, individuals with DS of all ages are less at risk from hypertension than their peers in the general population (incidence rate ratio 0.3, 95% CI 0.3 to 0.4)71. Although hypertension has not (yet) been clearly related to CAA-ICH in DS and in fact gives rise to a different pattern of haemorrhage, it is theoretically possible that higher blood pressure may increase the likelihood of aneurism and bleeds in vessels severely affected by CAA in those with dup-APP compared with individuals with DS.\n\n\nFurther research\n\nFurther epidemiological data are needed in DS regarding haemorrhagic stroke—a diagnostic category, not a single entity. CAA-ICH can be distinguished from other forms of haemorrhagic stroke on clinical grounds by using the validated Boston criteria4. The age-related risk for CAA-ICH in DS should be explored using susceptibility weighted imaging magnetic resonance imaging scans to detect microbleeds, which will allow comparison against dup-APP and sporadic AD to confirm relative burden of disease in these groups.\n\nThe role of factors involved in the clearance of Aβ in DS should be explored in more depth, as this could help to reveal potential drug targets to reduce CAA and associated ICH. Specifically, we have identified gaps in knowledge of the relationship between Aβ 40/Aβ 42 ratios and development of CAA and ICH in DS, on one hand, and Aβ clearance by endocytosis, enzymatic degradation, and removal through the BBB, on the other. Furthermore, direct comparison between DS and dup-APP cases and models is required. In this regard, mouse modelling with partial triplication of areas of chromosome 21 might identify an area of the chromosome that modulates the risk of CAA-ICH. CAA develops in several AD mouse models, including the Tg2576 (APP expressed under the PrP promoter)72, and J20 mouse model (APP transgenics with Swedish and Indiana mutations), particularly after 11 to 12 months of age73,74. The Tg-SwDI mouse is the most widely used model for studying CAA, containing the Swedish, Dutch, and Iowa mutations and developing CAA at 6 months75. Therefore, different partial trisomy strains could be crossed to transgenic mice expressing forms of APP that give rise to CAA and micro-haemorrhages; double mutant progeny could be assessed for CAA to see whether regions of chromosome 21 mediate increased or reduced pathology compared with mice carrying the APP transgene alone. If such a region were found, then it would give us dosage-sensitive candidate genes affecting the risk of CAA-ICH. Table 2 summarises some of the important unanswered questions generated by this review.\n\nAβ, amyloid-beta; AD, Alzheimer’s disease; APOE, apolipoprotein E; APP, amyloid precursor protein; CAA, cerebral amyloid angiopathy; DS, Down syndrome; dup-APP, duplication (i.e. having three copies) of the amyloid precursor protein region on chromosome 21; ICH, intracerebral haemorrhage; SOD, superoxide dismutase.\n\n\nConclusions\n\nThere is much variation between individuals with DS and development of clinical dementia and associated CAA and ICH. Variability in phenotypic and pathological expression, however, is not unique to DS but has also been reported in familial AD, suggesting the presence of genetic and non-genetic factors with disease-modifying effects. Intriguingly, individuals with DS appear to have much lower risk for developing ICH as well as some indications of less severe CAA when compared with families with dup-APP, despite also having three copies of the APP gene, suggesting that other genes on chromosome 21 may provide some protection against the effects of APP overdose. This review of the literature suggests that this lower prevalence and seemingly protective effect of trisomy 21 may be related to a difference in the clearance of Aβ, although other factors such as neuroinflammation, atherosclerosis, oxidative stress, and lower blood pressure could also have a role. Insights into these factors may provide important information about mechanisms of disease, which can be exploited to identify treatment strategies. For example, if it turns out that low blood pressure helps to protect individuals with DS from ICH, then that would suggest an important strategy to offer individuals from families with familial AD mutations at risk for CAA and ICH.",
"appendix": "Competing interests\n\n\n\nAndré Strydom has acted as an investigator in clinical trials of medications for Down syndrome sponsored by Roche Pharmaceuticals.\n\n\nGrant information\n\nThis work was funded by Wellcome Trust Strategic Grant No. 098330/Z/12/Z conferred upon The LonDownS Consortium UK. Dean Nizetic is funded also by the Lee Kong Chian School of Medicine, Nanyang Technological University-Singapore Start-up Grant.\n\n\nReferences\n\nHolland AJ, Hon J, Huppert FA, et al.: Incidence and course of dementia in people with Down's syndrome: findings from a population-based study. J Intellect Disabil Res. 2000; 44(Pt 2): 138–46. PubMed Abstract | Publisher Full Text\n\nVinters HV, Gilbert JJ: Cerebral amyloid angiopathy: incidence and complications in the aging brain. II. The distribution of amyloid vascular changes. Stroke. 1983; 14(6): 924–8. 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PubMed Abstract | Publisher Full Text\n\nLemere CA, Blusztajn JK, Yamaguchi H, et al.: Sequence of deposition of heterogeneous amyloid beta-peptides and APO E in Down syndrome: implications for initial events in amyloid plaque formation. Neurobiol Dis. 1996; 3(1): 16–32. PubMed Abstract | Publisher Full Text\n\nMotte J, Williams RS: Age-related changes in the density and morphology of plaques and neurofibrillary tangles in Down syndrome brain. Acta Neuropathol. 1989; 77(5): 535–46. PubMed Abstract | Publisher Full Text\n\nDe Jonghe C, Esselens C, Kumar-Singh S, et al.: Pathogenic APP mutations near the gamma-secretase cleavage site differentially affect Abeta secretion and APP C-terminal fragment stability. Hum Mol Genet. 2001; 10(16): 1665–71. PubMed Abstract | Publisher Full Text\n\nHerzig MC, Winkler DT, Burgermeister P, et al.: Abeta is targeted to the vasculature in a mouse model of hereditary cerebral hemorrhage with amyloidosis. Nat Neurosci. 2004; 7(9): 954–60. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMoore S, Evans LD, Andersson T, et al.: APP metabolism regulates tau proteostasis in human cerebral cortex neurons. Cell Rep. 2015; 11(5): 689–96. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIsrael MA, Yuan SH, Bardy C, et al.: Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells. Nature. 2012; 482(7384): 216–20. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nShi Y, Kirwan P, Smith J, et al.: A human stem cell model of early Alzheimer's disease pathology in Down syndrome. Sci Transl Med. 2012; 4(124): 124ra29. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMurray A, Letourneau A, Canzonetta C, et al.: Brief report: isogenic induced pluripotent stem cell lines from an adult with mosaic down syndrome model accelerated neuronal ageing and neurodegeneration. Stem Cells. 2015; 33(6): 2077–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee CY, Landreth GE: The role of microglia in amyloid clearance from the AD brain. J Neural Transm (Vienna). 2010; 117(8): 949–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSelkoe DJ: Clearing the brain's amyloid cobwebs. Neuron. 2001; 32(2): 177–80. PubMed Abstract | Publisher Full Text\n\nShibata M, Yamada S, Kumar SR, et al.: Clearance of Alzheimer's amyloid-ss1-40 peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier. J Clin Invest. 2000; 106(12): 1489–99. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeller RO, Djuanda E, Yow HY, et al.: Lymphatic drainage of the brain and the pathophysiology of neurological disease. Acta Neuropathol. 2009; 117(1): 1–14. PubMed Abstract | Publisher Full Text\n\nPortelius E, Soininen H, Andreasson U, et al.: Exploring Alzheimer molecular pathology in Down's syndrome cerebrospinal fluid. Neurodegener Dis. 2014; 14(2): 98–106. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWilcock DM, Hurban J, Helman AM, et al.: Down syndrome individuals with Alzheimer's disease have a distinct neuroinflammatory phenotype compared to sporadic Alzheimer's disease. Neurobiol Aging. 2015; 36(9): 2468–74. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSudduth TL, Greenstein A, Wilcock DM: Intracranial injection of Gammagard, a human IVIg, modulates the inflammatory response of the brain and lowers Aβ in APP/PS1 mice along a different time course than anti-Aβ antibodies. J Neurosci. 2013; 33(23): 9684–92. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTarasoff-Conway JM, Carare RO, Osorio RS, et al.: Clearance systems in the brain-implications for Alzheimer disease. Nat Rev Neurol. 2015; 11(8): 457–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKanekiyo T, Liu CC, Shinohara M, et al.: LRP1 in brain vascular smooth muscle cells mediates local clearance of Alzheimer's amyloid-β. J Neurosci. 2012; 32(46): 16458–65. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDeane R, Zlokovic BV: Role of the blood-brain barrier in the pathogenesis of Alzheimer's disease. Curr Alzheimer Res. 2007; 4(2): 191–7. PubMed Abstract | Publisher Full Text\n\nCheung C, Goh YT, Zhang J, et al.: Modeling cerebrovascular pathophysiology in amyloid-β metabolism using neural-crest-derived smooth muscle cells. Cell Rep. 2014; 9(1): 391–401. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMiners JS, Baig S, Palmer J, et al.: Abeta-degrading enzymes in Alzheimer's disease. Brain Pathol. 2008; 18(2): 240–52. PubMed Abstract | Publisher Full Text\n\nCarpentier M, Robitaille Y, DesGroseillers L, et al.: Declining expression of neprilysin in Alzheimer disease vasculature: possible involvement in cerebral amyloid angiopathy. J Neuropathol Exp Neurol. 2002; 61(10): 849–56. PubMed Abstract | Publisher Full Text\n\nMiners JS, Van Helmond Z, Chalmers K, et al.: Decreased expression and activity of neprilysin in Alzheimer disease are associated with cerebral amyloid angiopathy. J Neuropathol Exp Neurol. 2006; 65(10): 1012–21. PubMed Abstract | Publisher Full Text\n\nYamada M, Sodeyama N, Itoh Y, et al.: Association of neprilysin polymorphism with cerebral amyloid angiopathy. J Neurol Neurosurg Psychiatr. 2003; 74(6): 749–51. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiners JS, Morris S, Love S, et al.: Accumulation of insoluble amyloid-β in down's syndrome is associated with increased BACE-1 and neprilysin activities. J Alzheimers Dis. 2011; 23(1): 101–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWang DS, Lipton RB, Katz MJ, et al.: Decreased neprilysin immunoreactivity in Alzheimer disease, but not in pathological aging. J Neuropathol Exp Neurol. 2005; 64(5): 378–85. PubMed Abstract | Publisher Full Text\n\nDeane R, Wu Z, Zlokovic BV: RAGE (yin) versus LRP (yang) balance regulates alzheimer amyloid beta-peptide clearance through transport across the blood-brain barrier. Stroke. 2004; 35(11 Suppl 1): 2628–31. PubMed Abstract | Publisher Full Text\n\nLott IT, Head E: Alzheimer disease and Down syndrome: factors in pathogenesis. Neurobiol Aging. 2005; 26(3): 383–9. PubMed Abstract | Publisher Full Text\n\nSchley D, Carare-Nnadi R, Please CP, et al.: Mechanisms to explain the reverse perivascular transport of solutes out of the brain. J Theor Biol. 2006; 238(4): 962–74. PubMed Abstract | Publisher Full Text\n\nWilcock DM, Colton CA: Immunotherapy, vascular pathology, and microhemorrhages in transgenic mice. CNS Neurol Disord Drug Targets. 2009; 8(1): 50–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZis P, Dickinson M, Shende S, et al.: Oxidative stress and memory decline in adults with Down syndrome: longitudinal study. J Alzheimers Dis. 2012; 31(2): 277–83. PubMed Abstract | Publisher Full Text\n\nZis P, McHugh P, McQuillin A, et al.: Memory decline in Down syndrome and its relationship to iPF2alpha, a urinary marker of oxidative stress. PLoS One. 2014; 9(6): e97709. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPark L, Wang G, Moore J, et al.: The key role of transient receptor potential melastatin-2 channels in amyloid-β-induced neurovascular dysfunction. Nat Commun. 2014; 5: 5318. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGreenberg SM, Rebeck GW, Vonsattel JP, et al.: Apolipoprotein E epsilon 4 and cerebral hemorrhage associated with amyloid angiopathy. Ann Neurol. 1995; 38(2): 254–9. PubMed Abstract | Publisher Full Text\n\nGreenberg SM, Briggs ME, Hyman BT, et al.: Apolipoprotein E epsilon 4 is associated with the presence and earlier onset of hemorrhage in cerebral amyloid angiopathy. Stroke. 1996; 27(8): 1333–7. PubMed Abstract | Publisher Full Text\n\nNicoll JA, Burnett C, Love S, et al.: High frequency of apolipoprotein E epsilon 2 allele in hemorrhage due to cerebral amyloid angiopathy. Ann Neurol. 1997; 41(6): 716–21. PubMed Abstract | Publisher Full Text\n\nPhillips MC: Molecular mechanisms of cellular cholesterol efflux. J Biol Chem. 2014; 289(35): 24020–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTansley GH, Burgess BL, Bryan MT, et al.: The cholesterol transporter ABCG1 modulates the subcellular distribution and proteolytic processing of beta-amyloid precursor protein. J Lipid Res. 2007; 48(5): 1022–34. PubMed Abstract | Publisher Full Text\n\nKim WS, Rahmanto AS, Kamili A, et al.: Role of ABCG1 and ABCA1 in regulation of neuronal cholesterol efflux to apolipoprotein E discs and suppression of amyloid-beta peptide generation. J Biol Chem. 2007; 282(5): 2851–61. PubMed Abstract | Publisher Full Text\n\nBurgess BL, Parkinson PF, Racke MM, et al.: ABCG1 influences the brain cholesterol biosynthetic pathway but does not affect amyloid precursor protein or apolipoprotein E metabolism in vivo. J Lipid Res. 2008; 49(6): 1254–67. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBoehm-Cagan A, Michaelson DM: Reversal of apoE4-driven brain pathology and behavioral deficits by bexarotene. J Neurosci. 2014; 34(21): 7293–301. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAlexander M, Petri H, Ding Y, et al.: Morbidity and medication in a large population of individuals with Down syndrome compared to the general population. Dev Med Child Neurol. 2016; 58(3): 246–54. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFaraco G, Park L, Zhou P, et al.: Hypertension enhances Aβ-induced neurovascular dysfunction, promotes β-secretase activity, and leads to amyloidogenic processing of APP. J Cereb Blood Flow Metab. 2015. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nThanopoulou K, Fragkouli A, Stylianopoulou F, et al.: Scavenger receptor class B type I (SR-BI) regulates perivascular macrophages and modifies amyloid pathology in an Alzheimer mouse model. Proc Natl Acad Sci U S A. 2010; 107(48): 20816–21. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTong XK, Nicolakakis N, Kocharyan A, et al.: Vascular remodeling versus amyloid beta-induced oxidative stress in the cerebrovascular dysfunctions associated with Alzheimer's disease. J Neurosci. 2005; 25(48): 11165–74. PubMed Abstract | Publisher Full Text\n\nDavis J, Xu F, Deane R, et al.: Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch/Iowa mutant form of amyloid beta-protein precursor. J Biol Chem. 2004; 279(19): 20296–306. PubMed Abstract | Publisher Full Text\n\nRemes AM, Finnilä S, Mononen H, et al.: Hereditary dementia with intracerebral hemorrhages and cerebral amyloid angiopathy. Neurology. 2004; 63(2): 234–40. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13802",
"date": "12 May 2016",
"name": "Ira Lott",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13803",
"date": "12 May 2016",
"name": "Juan Fortea",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-876
|
https://f1000research.com/articles/5-875/v1
|
12 May 16
|
{
"type": "Case Report",
"title": "Case Report: Cervical chondrocalcinosis as a complication of Gitelman syndrome",
"authors": [
"Zahra Iqbal",
"Paul Mead",
"John A. Sayer",
"Zahra Iqbal",
"Paul Mead"
],
"abstract": "Gitelman syndrome is an inherited tubulopathy leading to a hypokalaemic metabolic alkalosis with hypomagnesaemia and hypocalciuria. Most cases are due to mutations in SLC12A3, encoding the apical thiazide sensitive co-transporter in the distal convoluted tubule. Musculoskeletal effects of Gitelman syndrome are common, including muscle weakness, tetany and cramps. Chronic hypomagnesaemia can lead to chondrocalcinosis, which often affects knees but can affect other joints. Here we present a case of Gitelman syndrome complicated by cervical chondrocalcinosis leading to neck pain and numbness of the fingers. Treatments directed at correcting both hypokalaemia and hypomagnesaemia were initiated and allowed conservative non-surgical management of the neck pain. Recognition of chondrocalcinosis is important and treatments must be individualised to correct the underlying hypomagnesaemia.",
"keywords": [
"chondrocalcinosis",
"Gitelman syndrome",
"hypomagnesaemia",
"musculoskeletal",
"SLC12A3"
],
"content": "Background\n\nGitelman syndrome (GS) is an autosomal recessive tubulopathy due to mutations in SLC12A3 encoding the thiazide sensitive sodium chloride co-transporter (NCC) in the distal convoluted tubule. The estimated prevalence is 1:40,0001. Biochemically the phenotype is similar to long-term thiazide diuretic treatment: hypokalemia, hypomagnesemia, a hypochloraemic metabolic alkalosis and reduced urinary calcium levels2. Although an inherited condition, the disease is usually diagnosed during adolescence or early adult life. However presentations late in life, often with chondrocalcinosis do occur3,4.\n\n\nCase report\n\nA 55-year-old lady was referred to the renal unit with persistently low serum potassium and magnesium levels following an episode of acute cholecystitis. Urinary electrolytes confirmed potassium wasting and hypocalciuria. On admission, her serum electrolytes were deranged: potassium 2.5 mmol/L, magnesium 0.31 mmol/L, corrected calcium 2.04 mmol/L, sodium 134 mmol/L and creatinine 53 µmol/L. Additional biochemistry tests confirmed hyperreninaemic hyperaldosteronism (renin >14.4 pmol/ml/hr (NR 0.5–3.1) and aldosterone 2794 pmol/L (NR 100–800)). Random urine sodium was 97 mmol/l, urine potassium 33 mmol/l and urine osmolality 467 mosm/kg. Biochemically, the diagnosis was consistent with GS.\n\nShe previously had no other significant medical history but had required NSAIDs for longstanding back, hip and neck pain. She was commenced on oral potassium and magnesium supplements (magnesium oxide 16 mmol/day) together with spironolactone 100 mg daily as a long term treatment. Molecular genetic analysis confirmed GS with the identification of compound heterozygous mutations in SLC12A3 (p.Arg209Gln and p.Ser615Leu)5. Despite oral supplementation, serum magnesium levels remained low (0.5–0.6 mmol/L).\n\nAt 60 years of age, she had a MRI spine examination for worsening neck pain and the onset of numbness in her fingers. The MRI spine revealed widespread chondrocalcinosis in the cervical spine and soft tissues, with a large ossified bony bar at the level of C3 and C4 compressing the spinal cord (Figure 1). In addition, there were multiple areas of chondrocalcinosis in the intra-vertebral discs, annulus fibrosus, ligamentum flavum and in the transverse ligament behind the odontoid process. Despite the fingertip numbness and severe chondrocalcinosis, physical examination demonstrated no apparent neurological loss, with normal, tone, power, reflexes and sensation. Neurosurgical advice was sought and a conservative approach was adopted. Oral magnesium supplementation was changed to magnaspartate and increased to 40 mmol/day in an attempt to normalise serum magnesium levels and prevent progression of the chondrocalcinosis and improve symptoms. From 60 to 62 years of age the serum magnesium has been maintained at near normal levels (0.6–0.75 mmol/L) with improvement of musculoskeletal symptoms and no progression of any functional deficit in hand movements. Neurology follow-up continues to adopt observational and conservative management.\n\nThere are also multiple areas of calcification involving the intravertebral discs, annulus fibrosis, the ligmentum flavum and the transverse ligament behind the odontoid process.\n\n\nDiscussion\n\nChondrocalcinosis is the deposition of calcium pyrophosphate crystals in the articular cartilages throughout the body and has been associated with the longstanding hypomagnesaemia secondary to GS6. Chondrocalcinosis may cause swelling, heat and tenderness over the affected joints. As well as GS, chondrocalcinosis may also be seen in association with hyperparathyroidism, haemochromatosis and hypophosphatasia.\n\nChondrocalcinosis is a known complication of GS and can affect various joints, most typically knees7. Cervical spine chondrocalcinosis due to GS, however, is not often reported. Calcium pyrophosphate dehydrate deposits in the peri-odontoid soft tissues can lead to a condition called ‘crowned dens syndrome’ which causes acute neck pain and has been associated with GS8. Treatment relies on magnesium replacement and symptom control with non-steroidal anti-inflammatory drugs. Surgery is rarely performed.\n\nPatients with GS can experience salt craving, tetany and cramps, fatigue and severe lethargy, often impacting greatly on their quality of life5. The chondrocalcinosis associated with it further adds to the musculoskeletal disease burden. It is therefore important to monitor patients and try to correct the potassium and magnesium disturbances to prevent acute exacerbations and progression9. Complete normalisation of serum magnesium is often difficult due to diarrhoea associated with magnesium supplements. Various magnesium preparations are available, including magnesium oxide, magnesium glycerophosphate, magnaspartate, and magnesium lactate (Mag-Tab SR), which is slow release and is often better tolerated. Trial of various preparations and individual tailoring of dosing is required.\n\nIn summary we present a case of GS presenting with typical electrolyte disturbances (hypokalaemic metabolic alkalosis and hypomagnesaemia) complicated by severe chrondrocalcinosis of the cervical spine. Treatment directed at correcting these electrolyte disturbances has allowed an improvement of symptoms and avoidance of neurosurgery.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for publication of this case report and any accompanying images and/or other details that could potentially reveal the patient’s identity.",
"appendix": "Author contributions\n\n\n\nJAS conceived the idea. ZI, PM and JAS wrote the article and approved the final version.\n\n\nCompeting interests\n\n\n\nThe authors have no competing interests to declare.\n\n\nGrant information\n\nJAS is supported by the Northern Counties Kidney Research Fund and the Medical Research Council (MR/M012212/1). The authors have no conflicts of interest to declare.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank the patient for contributing to the study.\n\n\nReferences\n\nKnoers NV, Levtchenko EN: Gitelman syndrome. Orphanet J Rare Dis. 2008; 3: 22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSayer JA, Pearce SH: Diagnosis and clinical biochemistry of inherited tubulopathies. Ann Clin Biochem. 2001; 38(Pt 5): 459–70. PubMed Abstract | Publisher Full Text\n\nBrambilla G, Perotti M, Perra S, et al.: It is never too late for a genetic disease: a case of a 79-year-old man with persistent hypokalemia. J Nephrol. 2013; 26(3): 594–8. PubMed Abstract\n\nEa HK, Blanchard A, Dougados M, et al.: Chondrocalcinosis secondary to hypomagnesemia in Gitelman's syndrome. J Rheumatol. 2005; 32(9): 1840–2. PubMed Abstract\n\nCruz DN, Shaer AJ, Bia MJ, et al.: Gitelman's syndrome revisited: an evaluation of symptoms and health-related quality of life. Kidney Int. 2001; 59(2): 710–7. PubMed Abstract | Publisher Full Text\n\nRichette P, Ayoub G, Lahalle S, et al.: Hypomagnesemia associated with chondrocalcinosis: a cross-sectional study. Arthritis Rheum. 2007; 57(8): 1496–501. PubMed Abstract | Publisher Full Text\n\nIqbal Z, Sayer JA: Chondrocalcinosis and Gitelman syndrome. QJM. 2016; pii: hcw045. PubMed Abstract | Publisher Full Text\n\nGutierrez M, Silveri F, Bertolazzi C, et al.: Gitelman syndrome, calcium pyrophosphate dihydrate deposition disease and crowned dens syndrome. A new association? Rheumatology (Oxford). 2010; 49(3): 610–3. PubMed Abstract | Publisher Full Text\n\nCalò L, Punzi L, Semplicini A: Hypomagnesemia and chondrocalcinosis in Bartter's and Gitelman's syndrome: review of the pathogenetic mechanisms. Am J Nephrol. 2000; 20(5): 347–50. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13809",
"date": "16 May 2016",
"name": "Coralie Bingham",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 about an unusual complication of a rare disease. The genetic background to this disorder is covered. I think this report will be useful to other clinicians who may rarely see a case of Gitelman syndrome. There is a clear discussion of the medical management strategy used in this patient which lead to an improvement of symptoms and the avoidance of surgery. I think this report adds to knowledge about this rare disease and merits indexation.",
"responses": []
},
{
"id": "14146",
"date": "03 Jun 2016",
"name": "Nicholas G Larkins",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 article describing an interesting presentation of an uncommon condition. The case would be useful to other clinicians, as there is some important discussion of magnesium preparations.\n\nI note the authors reference another case report published this year by themselves of a 37 year female with chonedrocalcinosis and GS. These could have been combined, with a more substantial discussion for a more useful contribution to the literature.",
"responses": []
},
{
"id": "13933",
"date": "13 Jun 2016",
"name": "Andrew Mallett",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 constructed and executed Case Report which is informative both with regards to the uncommon complication and the primary inherited kidney disease it pertains to. It is likely to be of interest and utility for clinicians who may encounter a patient with Gitelman Syndrome. The background, case history and discussion are cohesive, add to the knowledge base and are important for advancing the understanding and clinical management of this rare disease.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-875
|
https://f1000research.com/articles/5-870/v1
|
11 May 16
|
{
"type": "Research Article",
"title": "Reproductive success is predicted by social dynamics and kinship in managed animal populations",
"authors": [
"Saul J. Newman",
"Simon Eyre",
"Catherine H. Kimble",
"Mauricio Arcos-Burgos",
"Carolyn J. Hogg",
"Simon Easteal",
"Simon Eyre",
"Catherine H. Kimble",
"Mauricio Arcos-Burgos",
"Carolyn J. Hogg",
"Simon Easteal"
],
"abstract": "Kin and group interactions are important determinants of reproductive success in many species. Their optimization could, therefore, potentially improve the productivity and breeding success of managed populations used for agricultural and conservation purposes. Here we demonstrate this potential using a novel approach to measure and predict the effect of kin and group dynamics on reproductive output in a well-known species, the meerkat Suricata suricatta. Variation in social dynamics predicts 30% of the individual variation in reproductive success of this species in managed populations, and accurately forecasts reproductive output at least two years into the future. Optimization of social dynamics in captive meerkat populations doubles their projected reproductive output. These results demonstrate the utility of a quantitative approach to breeding programs informed by social and kinship dynamics. They suggest that this approach has great potential for improvements in the management of social endangered and agricultural species.",
"keywords": [
"Conservation",
"social interaction",
"kin selection",
"meerkats",
"group dynamics"
],
"content": "Introduction\n\nThe growing global crisis in biodiversity has caused the survival of endangered species to become increasingly dependent on managed breeding programs. These are generally designed to maximize reproductive success1,2, but they sometimes fail to increase or even maintain population sizes1. Similar pressures to increase reproductive success in managed social species exist in agriculture, where the maximization of reproductive output is often synonymous with increasing yield.\n\nA possible reason for poor or underperforming outcomes is that kinship and group structures, which are known to affect breeding success in wild populations3–6, are suboptimal in managed populations. Therefore, it may be possible to improve the success of conservation and agricultural breeding programs involving social species by optimizing kin and group interactions.\n\nHere we investigate this possibility using kin and group interactions inferred from detailed longitudinal records of kinship, migration between institutions and reproduction records maintained by zoological institutions. We apply novel methods to predict reproductive success in populations of the highly social meerkat, Suricata suricatta, from observed variation in age, sex, relatedness and social group structure.\n\nMeerkats are an important model species in the development of evolutionary theory, providing an empirical test case for hypotheses about social evolution derived from both game and kin selection theory7–9. Game theory predicts that individuals will interact cooperatively or competitively to maximize payoffs in direct fitness10, and that these interactions do not depend on kinship structure. In contrast, kin selection theory predicts that individuals will seek to maximize their inclusive fitness by modifying the direct fitness of biological kin11,12, implying that the fitness effects of social interactions between cohabiting individuals are significantly affected by kinship structure.\n\nTesting the alternative predictions of these theories requires longitudinal life-history data on social dynamics, reproductive success, survival, and kinship structure that are difficult to obtain from wild populations, but which are available for some managed populations in zoos. Here we evaluate and compare the predictions of these two theoretical approaches with data for managed meerkat populations.\n\nWe obtained data for managed meerkat populations in North America and Australasia from zoological studbooks, which record extensive life history data about individual animals to a time resolution of one day across multiple decades. These genealogies were documented to avoid inbreeding, and records span more than 113 years, with comprehensive coverage across the last 50 years. Across this period there is complete documentation of location of > 99% of individuals in the population, the exact dates of their transfer between institutions, and of their birth and death dates.\n\nMeerkat life histories can be accurately reconstructed on an individual level from these data. Interactions between managed meerkats occur in a finite, discrete geographic space: individuals either have the possibility of interacting within the same zoo in either breeding or non-breeding enclosures, or they are completely socially isolated from one another in separate enclosures. Coupled with detailed genealogical data, this property can be used to reconstruct detailed individual life histories of individual captive meerkats, including the number, sex and relatedness of all other individuals living in the same zoo.\n\nTo quantify kinship in the complex and inbred multigenerational meerkat populations for which data are available, we initially mapped the relatedness of individuals in the populations as directed acyclic graphs of kinship (kDAGs), in which nodes represent individuals, and parents are connected to their children by directed edges (Figure 1; Figure S1). We then evaluated standard approaches to estimating relatedness. All zoo-bred individuals with known parentage were included in our analysis.\n\n(A) A section of the kinship-directed acyclic graph (kDAG) in Figure S1. (B) Within the kDAG, nodes representing parents are connected to nodes representing their children by directed edges. (C) Each individual in the graph is related to a finite sub-graph of individuals (coefficient of relatedness, r, indicated by shading in C–E). Even at small diameters, sub-graph structures can be highly variable (as between C and D). However all possible sub-graphs of a kDAG can be reduced to a finite set of paths with nodes grouped into ascendant (I), lateral (II) or descendant (III) classes (E), without any loss of information on inheritance patterns or relatedness. This reduced set of paths is represented as a SIG (Figure S5) in which nodes connected by ascendant paths (I) can include only one individual of each sex, and nodes in lateral (II) and descendant (III) SIG paths can contain any number of individuals of any sex.\n\n\nMaterials and methods\n\nExtensive demographic, geographic and genealogical data were obtained for two large independent managed meerkat populations, the North American Regional Meerkat Studbook (21; n = 2843) and the Australasian Regional Meerkat Studbook (22; n = 668). Within these populations, 2083 individuals (59%) have two documented parents and 973 (28%) have four documented grandparents. Of 1807 records of transfer between zoological institutions, 89% are accurate to the exact day, and less than 1 per cent of all individuals have been lost to follow-up. Our analysis was based only on records of meerkats living in zoos. We excluded records from other organizations such as animal suppliers and private owners, or institutions that did not have listed addresses (n = 23), which we recoded as ‘wild’. Entry of individual meerkats to zoos from these other institutions was treated as equivalent to entry from wild populations. No information about relationships among individuals at the data-collection baseline is available, and individuals entering the population (n = 185) were treated as unrelated. Individuals were also excluded from analysis if they had been lost to follow-up or released into the wild or wild-encoded institutions (n=237).\n\nBirth, death and transfer dates were encoded numerically and rounded to the nearest month. We excluded dates that were not recorded to a time resolution of one month or for which uncertainty was indicated in the records (e.g., “1988” or “~Mar 1988”; ~13% of recorded dates).\n\nThe kinship structures of the managed meerkat populations were represented as kinship directed acyclic graphs (kDAGs; Figure S1) in which each node represents an individual connected to its offspring by a directed edge (Figure S1A).\n\nThe extent and complexity of a meerkat’s kinship network changes over time and there is a general increase in the connectedness and complexity of the overall meerkat kDAG, as indicated by increased density of directed edges (Figure S2A), and by the closeness centrality23 of nodes (Figure S2B). The addition of immigrant meerkats introduces variation in the rate and direction of these changes.\n\nThe way genealogical information has been collected over time can bias results from traditional kinship measures. Estimating Wright’s coefficient of relatedness r13 in our genealogy using the “kinship2” package24 in the R environment version 3.1.225, we found that the mean coefficient of relatedness between an individual and the general population (rmean) was dependent on: order of entry into the genealogy, number of documented ancestors within the zoo system, and number of higher-degree relatives (Figure S3).\n\nRelatedness coefficients between kinship categories such as “mother” or “second cousin” are highly dependent on the degree of genealogical connectedness and completeness. Most mother-child pairs are related by exactly r = 0.5 when the genealogy is small. However, mother-child pairs may also be related by many other kinship categories, such as second and fourth cousins. As the number of individuals in the kDAG grows, higher-degree relationships became more completely documented, and an increasing number of mother-daughter pairs became connected by other relationship paths.\n\nThis accumulation of higher-degree relatedness continued until 39% of all mother-daughter pairs had coefficients of relatedness > 0.5 (Figure S3C). As the kDAG size increases, an increasing number of individuals have a unique degree of inter-relatedness (Figure S3B). The problem is more pronounced between individuals with primary connections through higher degree kinship categories such as “cousin” (Figure S3D).\n\nTo overcome these biases we developed the standard inheritance graph (SIG) as a new method of quantifying kinship in these large genealogies.\n\nEvery individual within a non-hermaphroditic, exclusively sexual population has exactly two parents of opposite sex. The number of edge paths connecting any individual to their parents is fixed: every individual in a sexual population is connected to exactly one female parent by exactly one ascendant “maternal” edge, and one male parent by one ascendant “paternal” edge.\n\nIn a population of selfing hermaphrodites, all individuals in a population still have exactly one father and exactly one mother, but these “parents” may be the same organism. The number of parents is variable, but the number of paths connecting an individual to its ascendant kin is constant: individuals have exactly one ascendant maternal path and exactly one ascendant paternal path, which terminate at exactly one female and exactly one male, respectively. The set of paths connecting a focal individual to its first-degree ascendants is the same whether both parents are different individuals or the same individual.\n\nThis invariant set of paths connecting a focal individual to a variable number of ascendants, exists at all higher degrees of relatedness (Figure S4). An individual may have only two grandparents in an inbred lineage, but they still possess exactly four ascendant grandparental paths: a path of two ascendant male edges to their father’s father; a path of two ascendant female edges to their mother’s mother; a path of one ascendant female edge followed by one ascendant male edge to their mother’s father; and a path of one ascendant male edge followed by one ascendant edge to their father’s mother.\n\nThis underlying structure extends to all genealogies with exactly two sexes. If all ascendant and non-ascendant paths are documented, in any genealogy of any size, the resultant set of paths falls into a single fixed set of possible paths (Figure S4C, Figure S5). In addition, all ascendant relatives at φ degrees of relatedness are connected to a focal individual by exactly 2φ unique edge paths (Figure S4B), and each ascendant path terminates at exactly one individual of a determinate sex.\n\nThe set of all ascendant and non-ascendant kinship informative paths has several useful qualities.\n\n1). Each path makes a fixed contribution to the total coefficient of relatedness between two individuals. If an individual is connected to a relative by a path of length φ parent-child edges, this path contributes kφ to the total coefficient of relationship between these two individuals, where k is the coefficient of relatedness between a parent and child.\n\n2). The coefficients of relatedness of different paths linking the same two individuals are additive. When and individual is connected to a relative by more than one path (e.g., when the same individual is both their maternal and paternal grandmother) the total coefficient of relatedness between them is equal to the sum of the coefficients of relatedness for all paths that connect them.\n\n3). Every path is informative of structural relatedness, independent of variation in coefficients of relatedness. For instance, both a maternal and paternal ascendant path of length 1 terminates at an equally related relative, a ‘mother’ and ‘father’, who are related to the focal individual by r = k. However, these equally related relatives are structurally different classes in the ascendant path set.\n\nPedigrees typically reached at least one wild founder within 4 ascendant generations in the meerkat populations, limiting the number of higher degree relationship paths that could be reliably observed between individuals. In turn, this limited the diameter of completely documented SIGs below 4 degrees of relatedness. We therefore selected all ‘focal’ individuals in the managed populations with a full set of documented first, second or third degree ascendant relatives using a simple pattern-matching algorithm in R 3.1.2 (25; Supplementary material). We constructed 1, 2 or 3-degree ascendant SIGs for each of these focal individuals, using the direct descendants the focal individual and their ancestors.\n\nEach focal individual’s reproductive success was estimated as the sum of relationship coefficients (rIII) to all direct descendent relatives over time (represented in the ‘III’ paths of the SIG in Figure 1F and Figure S5). For example, the reproductive success of a focal individual producing one child (r = 0.5) and one grand-child (r = 0.25) over the period n…n+1 is:\n\n∑nn+1rIII=0.75,\n\nBy including multiple generations of descendent relatives, this metric accounts for differential reproductive success among immediate offspring, and avoids weighting the production of sterile and fecund offspring equally.\n\nIn our analysis, the number of predictor variables was high relative to the number of cases being predicted. Group size and kinship metrics were further constrained in that they included mixed binary (e.g., the presence/absence of mothers), discrete and continuous variables that did not generally have simple distributions. Predictive algorithms built using parametric approaches, such as logistic regression, perform poorly under these conditions26.\n\nWe therefore constructed several recursively partitioned regression models (RPRMs) to predict reproductive success, using different sets of training variables. Built on a nonparametric learning algorithm, RPRMs can produce robust predictive models using large sets of variables of mixed data types. RPRMs are a robust data-driven technique of model construction26,27 that make no assumptions about the linearity of relationships and which are independent of variable ordering.\n\nRPRMs begin by finding the value of a predictor variable that best partitions cases of the outcome variable (the ‘parent node’) into two smaller populations or ‘child nodes’. This ‘best’ partition is found by identifying all possible partitions of all variables, calculating the sum of squares between child nodes using a simple analysis of variance for each partition, and selecting the partition that leads to the maximum difference in the between-node sum of squares.\n\nEach child node from this ‘best’ partition is then used as the parent node for a new round of partitioning. This splitting algorithm is applied recursively to all child nodes until one of three stopping criteria is satisfied. The splitting algorithm halts if a child node reaches a minimum sample size (the ‘minbucket’ parameter), a parent nodes contains too few cases to attempt a split (the ‘minsplit’ parameter), or when a split is unable to increase the fit of the model by a given factor (the ‘complexity parameter’).\n\nIn the absence of strict stopping criteria, this algorithm produces an over-fitted predictive model, represented by a binary decision tree of splitting criteria. We eliminated over-fitting in this model by pruning this decision tree using random-sampled cross validation.\n\nTo perform k-fold cross validation, the data were randomly subdivided into equally sized groups G1…Gk. For each successive group G1…Gk, the ith group Gi was excluded and an RPRM was constructed from the remaining data. All sub-trees of this model were then used to predict the outcome variable of the excluded Gi group. The accuracy of each sub-tree was measured as the R2 coefficient between predicted and observed values. After testing across all k groups, the sub-tree with the highest mean accuracy at predicting the outcome variable was selected out of all tested sub-trees. This selected binary tree represents the predictor most robust to variation introduced by sampling of the original data, and forms the output RPRM. Pruning RPRMs in this way effectively eliminates model over-fitting, returning a tree that robustly predicts the outcome variable26,28.\n\nTo further exclude the possibility of over-fitting, we externally cross-validated each RPRM in the Australasian meerkat data, data that was not used in model construction. The accuracy of each RPRM in predicting this external holdout population was calculated by measuring the correlation coefficient between the observed reproductive success in the Australasian meerkat population, and the reproductive success independently predicted by each RPRM in the North American population (Table 1).\n\nRPRMs were constructed using the ‘rpart’ package version 4.1-1 in R 3.1.225, using data from the North American meerkat population only21. We used identical parameters for all RPRMs, splitting using the ‘anova’ method, with minsplit and minbucket values of n=15, and the default complexity parameter of 0.01. All models were pruned using internal 10-fold cross-validation.\n\nAll RPRMs were constructed using a base set of predictor variables, including individual age and sex, and the age-specific female, male and total group size within each zoo. Added to these predictor variables were each of the variables quantifying the number of real present relatives, the number of living absent relatives, the number of global relatives, and the number of pseudo-kin, for four initial predictive models.\n\nAll models were trained and tested using focal individuals with complete 1, 2 or 3-degree SIGs, producing twelve predictive models (‘local kin’, ‘non-local kin’, ‘global kin’ and ‘pseudo kin’ predictive models, across 1, 2 and 3-degree SIGs; Table 1).\n\nTwo subsequent models were constructed from the local kin and pseudo kin variables. The ‘Split’ model retained the distinction between kin and non-kin by treating pseudo kin and local kin as independent variables. The ‘sigma’ model was constructed using identical data, but eliminated the distinction between kin and non-kin by summing local kin and pseudo kin to form a new, combined kinship class.\n\nAll of these models were constructed using the North American meerkat population data and tested, using identical parameters, in the independent Australasian meerkat population.\n\nWe developed a combined genetic/packing algorithm that optimizes social group structure to increase the reproductive success of the meerkat populations under realistic constraints (Figure S4). The aim was to predict how rearranging individuals and their relatives across zoos improved per capita reproductive success of the entire meerkat population.\n\nThe genetic/packing algorithm operated by generating 1000 seed populations by randomly assigning new locations, with replacement, to individuals. Seed populations were selected that did not exceed historic group sizes observed within a zoo over the past decade, and which did not reduce a zoo populations below thresholds shown in Table S1. These requirements formed simple packing constraints imposed in each randomization step of this algorithm.\n\nA genetic optimization algorithm was applied to each seed population. Five “mutated” populations were generated for each seed population, by randomly sampling (with replacement) groups of 50 individuals from each seed population and randomly reassigning them to new locations. These five mutated populations were tested for adherence to the inbuilt group size (packing) constraints. If populations failed to satisfy these constraints, the mutation process was repeated until five randomized populations were obtained that could be accommodated within existing zoo enclosures.\n\nThe population-wide reproductive success of each of the five mutated populations (plus the original seed population) was then predicted under these new conditions using the ‘sigma’ predictive model. The algorithm selected the population rearrangement with the highest predicted reproductive success across a 12-month period under this model.\n\nThis selected population was then used as the starting point for another identical round of mutation and selection. This entire process, of random population rearrangement within packing constraints, prediction of reproductive success under the new arrangement, and selection of the optimal population structure, was iterated 40 times for each of the initial 1000 seed populations (Figure S6). We then selected the population structure with the highest predicted population-wide reproductive success over 12 months, under the ‘sigma’ model predictions (Figure S6).\n\nSame-sex siblings born into single paternity litters have perfectly congruent family trees. Furthermore, these individuals have identical ages, and often occupy the same enclosures: these ‘littermates’ therefore often share exactly identical kinship and social structures.\n\nWe used this property of same-sex littermates to test the associations detected using the local kin and split models, where the best predictors of reproductive output were the numbers of cohabiting paternal and maternal siblings (Figure 2A).\n\n(A–C) SIG classes are represented as a ‘crane-fly’ plot for local kin, non-local kin, and pseudo-kin models respectively, with number of individuals in each SIG class indicated by node diameter, and predictive effect indicated by gray scale. (D) Observed changes in reproductive success after co-transferring same sex ‘littermates’ or unrelated ‘pseudo-siblings’ between institutions. Effect sizes are scaled independently in A–C.\n\nIndividuals in both meerkat populations are routinely transferred between institutions, in groups of variable composition. We selected all individuals sent to new institutions within groups of more than one individual (a ‘co-transfer’ group). Individuals known to have reproduced before the date of first transfer were excluded to eliminate any bias against transferring highly reproductive individuals. We then compared the effect on reproductive output of co-transfer with littermates and co-transfer with unrelated individuals of similar age and sex using a Kruskal-Wallis rank sum test29 in R version 3.1.225.\n\nGenetic measures of kinship, such as Wright’s coefficient of relatedness13, were affected by the stochastic arrival of unrelated individuals into the kDAG over time; failed to distinguish between equally related but distinct classes of kin; and exhibited complex edge effects (Figure S2, Figure S3). Relatedness coefficients also changed unpredictably over time in response to the changing connectedness and diameter of each kDAG, independent of actual changes in population structure (Figure S3).\n\nKinship classes, such as “mother” or “second cousin” provide an alternative approach. However, these also failed to capture the complexity of kinship structures in the meerkat populations because individuals can belong to more than one kinship class. As a result, the total number of kinship classes and the number of unique combinations of classes connecting biological relatives increase nonlinearly with increasing kDAG diameter. Even in small genealogies, almost all individuals are related to one another by rare non-equivalent combinations of kinship classes (Figure S3).\n\nTo overcome these limitations, we developed a new, unbiased approach to quantifying kinship based on ‘standard inheritance graphs’ (SIGs). This approach considerably reduced the complexity of genealogical networks while retaining all information on coefficients of relatedness and biological kinship structures.\n\nWe used the SIG approach to evaluate how kin- and non-kin-based social structure predicts reproductive success in the managed meerkat populations. We constructed a series of recursively partitioned regression models (RPRMs) to predict reproductive success from these variables, and the age and sex of the focal individual14.\n\nFor each month during the life of each focal individual, we constructed predictive models of reproductive success using several sets of predictor variables, using the number of: living relatives in each SIG class inhabiting the same zoo (local kin; α), living relatives in each SIG class inhabiting other zoos (non-local kin; β), living relatives in each SIG class, regardless of location (global kin; α + β), and unrelated individuals matching the age and sex of relatives in each SIG class that inhabited the same zoo (pseudo-kin; γ).\n\n\nResults\n\nVariation in local kin predicted ~19% of the individual variation in reproductive output in the Australasian meerkat population (Figure 2A, Table 1). Cohabitation with siblings and, to a lesser extent, uncles and aunts are the main contributors to this effect (Figure 2A). There is some contribution from focal individual age but none from either group size or focal individual sex. The ~8% of variation predicted by non-local kin (Table 1) follows a similar pattern (Figure 2B). The similar pattern arises because, where the number of individuals in a kinship category is constrained (e.g., the ‘mother’ category, which always contains just one individual), the presence of local kin implies a reciprocal absence of non-local kin. A ‘local kin’ effect is, therefore, mirrored by a reciprocal ‘non-local kin’ effect.\n\nThe predictive value of pseudo kin (mean r2 = 0.20) is similar to that of local kin. However, the two variables have distinct effects, as evident in the map of contributing model components (Figure 2C). Furthermore, when the two variables are included as separate variables in the ‘split’ model, their combined predictive value (mean r2 = 0.29) is substantially increased. In contrast, when kin and pseudo kin are combined as a single variable in the ‘sigma’ model their net predictive value (mean r2 = 0.09) is substantially less than when these variables are treated individually, indicating that kin and non-kin have distinct non-additive effects that partially cancel each other out when kinship structure is ignored (Table 1).\n\nPatterns of animal transfers between zoos also indicate distinct kin and non-kin effects on reproductive success. Variation in the number of siblings co-transferred to a new zoo is a strong predictor (p = 0.0002; Figure 2D) of reproductive success, with increased numbers of siblings associated with reduced reproductive output independent of litter size. In contrast, variation in the number of co-transferred non-siblings of the same sex and age does not predict subsequent reproductive success (p = 0.39; Figure 2D).\n\nHeritable variation in reproductive potential would result in a correlation between sibling number and subsequent reproductive success, which could confound these results. However, in these populations, when the effect of co-transfer is ignored, there is no correlation between sibling number and subsequent reproductive success (p = 0.30). Furthermore, co-transfer with increasing numbers of siblings significantly reduced reproductive success across litters of the same initial size.\n\nThe observed local-kin and non-local kin effects could also be affected by inter-kin correlations caused by heritable variation in reproductive potential, if there was a tendency for members of families with low reproductive potential to be co-located, and those from families with high reproductive potential to be located in different enclosures. If this were the case, however, global kin would have better predictive accuracy than either local kin or non-local kin, which is not what we observe. Furthermore, heritable effects could not explain how different relative classes with equal coefficients of relatedness have different predictive effects on reproduction (Figure 2A,B), or why the lifetime reproductive success of parents and offspring are not significantly correlated (p = 0.59).\n\nThus, distinct kin and non-kin effects can be identified. When these effects are treated separately, approximately 30% of variation in reproductive success in managed meerkat populations can be predicted from current social conditions.\n\nWe extended this analysis to investigate whether previous reproductive success and current social conditions could accurately predict future reproductive success over a number of months. We used the split model to capture both kin and non-kin effects and included previous reproductive success as a predictor variable. The outcome variable predicted by these models was the sum of future reproductive success at 24 monthly intervals, excluding all cases where the focal individual died during this period.\n\nAs before, RPRMs trained to predict future reproductive success in the North American population were tested in the independent Australasian population. The accuracy of these models, estimated as the correlation coefficient between predicted and observed values in the Australasian population, ranged from 0.21 to 0.40 over a two-year period (Figure 3). Distinct local kin and pseudo kin effects were again evident in these models, in addition to the relatively strong effects of previous reproductive success and group size.\n\nData on group and kinship structure in the North American meerkat population was used to train predictive models forecasting future reproductive success at monthly intervals up to 2 years. The predictive accuracy of these models was estimated as the correlation coefficient between predicted reproductive success based on the North American population and the reproductive success observed in the independent Australasian population (r2; 95% confidence intervals indicated by whiskers).\n\nThis result indicates that restructuring zoo populations to optimize their social composition could improve their future per capita reproductive success. We designed a recursive genetic optimization algorithm to explore this possibility further, taking account of the practical constraints of current zoo management practices (Figure S6).\n\nThe algorithm was applied to 1000 randomly generated starting population rearrangements of the 521 living North American meerkats housed in 89 zoos (Figure 4). Iterative optimization resulted in a general improvement in predicted 12-month reproductive output. The most optimal population configuration predicted a two-fold increase in the per capita reproductive success of the meerkat population compared to the baseline prediction (Figure 4), an increase made possible by a high level of variation in offspring number between individuals and zoos.\n\nThe resulting distribution of reproductive output scores are shown relative to the Holt-Winters (grey box) and recursively partitioned regression model (horizontal grey line) predictions for unaltered conditions (Figure S6).\n\n\nDiscussion\n\nThese results demonstrate that both kin-kin cohabitation and general social structure have independent value in predicting reproductive success. Our analysis supports a non-exclusive role for both game and inclusive fitness theory interactions, reconciling findings from different species that support either strong kin selection4,15 or game-theoretic interactions10,16 as important determinants of reproductive success.\n\nThe predictive accuracy of our models was possible because we detected differential interactions between equally related but distinct classes of kin using the SIG method. This enabled finer segregation of kinship classes than can be achieved by existing methods.\n\nThis finding has clear implications for the management of social species. Our analysis suggests the considerable scope for improving the success of managed breeding programs by moving individuals into optimally or near-optimally structured social groups, which can be achieved within the constraints of existing enclosures. For instance, the observation of reduced reproduction in the presence of siblings is supported by data on incest avoidance in wild meerkat populations9, and suggests a simple kinship-based mechanism for increasing reproductive success.\n\nWe developed this approach in the meerkat, a species of considerable scientific importance, but limited economic value. However, in principle the SIG approach is applicable to managed populations of any species of endangered or agricultural species exhibiting social behavior.\n\nMost agricultural animals were domesticated to take advantage of their social herding behaviors17, suggesting that there may be scope to improve their management through optimization of social or family structure. Some plant species display kin- and density-specific effects on survival, growth and productivity18–20, suggesting these traits might also be improved through optimization of kin structures in plants.\n\nKin-kin interactions within important agricultural species are often documented by large pedigrees from which high-degree kinship networks can be derived. We have focused here on reproductive success as an outcome, but this approach could be used to assess and modify the effect of social behavior on any trait from existing pedigree data, including agriculturally important traits such as growth rate and yield.\n\n\nData and software availability\n\nF1000Research: Dataset 1: De-identified meerkat data and code, 10.5256/f1000research.8713.d12167530\n\nDue to concerns over animal-rights based opposition to managing social and endangered species, locations and names have been de-identified. Identified data is available on request from the Zoo and Aquarium Association Australasia or the American Zoo and Aquarium Association.",
"appendix": "Author contributions\n\n\n\nSN designed the code, general statistical design, genetic packing and sorting algorithm, RPRM implementation and testing, and theoretical design of the study. SN and MAB contributed to the RPRM predictive algorithm design and implementation. SEy and CK provided both data and invaluable insight into the meerkat populations, and CH contributed to the design of realistic constraints for the packing/sorting algorithm. SE contributed to study design. SN and SE wrote the manuscript, with CH and MAB contributed to the editing, syntax and structure 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\nAcknowledgments\n\nWe thank Steinar Engen for his suggestion to analyze zoological pedigrees and the many volunteers, professional zookeepers and institutions that construct and maintain endangered and managed species studbooks, in particular the regional zoo and aquarium associations.\n\n\nSupplementary material\n\nSupplementary figures and table.\n\nEach individual is represented as a node and parent nodes are connected to offspring nodes by directed edges (A), for the North American (B–D) and Australasian (E–F) meerkat populations. Both graphs collapse to one major cluster (B, E), many smaller unconnected sub-graphs (C, F), and a set of unconnected nodes (D, G). Variation in birth dates is indicated by color gradients. Individuals with inaccurate (to month) or unknown dates of birth are shown as black nodes.\n\nBy sorting the kDAG radially by order of entry into the population (date of entry indicated by color in A; order of entry (N) indicated by radial position in A and B), change in graph connectedness is indicated by the increasing density of directed edges over time (A, grey edges) and the closeness centrality of nodes (B). The closeness centrality of a node is a measure of the mean path length required to reach all nodes in a graph from that node (16). Unlike other centrality measures, closeness centrality incorporates information from all paths between nodes, not just the shortest mean path. Higher closeness centrality indicates higher connectedness, with a baseline closeness centrality of zero for an unconnected node.\n\n(A) The mean coefficient of relatedness of focal individuals to the rest of the population is dependent on the order in which they enter the genealogy (either through birth or immigration) and is driven by changes in network connectedness and complex edge effects. (B) Individuals become increasingly connected by multiple relatedness paths as the diameter of the kinship network grows, increasing the number of unique values of r within each kinship category. (C) For instance, r between mother-child pairs increase from a single value (r=0.5) to 38 unique values as the kDAG increases in size. (D) A similar increase in unique r values occurs between paternal sibling pairs. Traditional kinship categories do not capture this variation in relatedness coefficients within the pedigree.\n\n(A) For any strictly 2-sex species, a genealogy of first-degree ascendants has only two possible states: an individual has either one mother and one father (A I), or one selfing hermaphroditic parent (A II). These two genealogies contain different numbers of individuals, related by different coefficients of relatedness, but they can be represented by identical set of “paths” linking parents of each sex to their offspring. In both cases a child has exactly one female parent and one male parent (as indicated within dashed-line box). The number of possible genealogies is therefore larger than the number of possible path combinations. (B) This relationship holds at higher degrees of relatedness. At two degrees of relatedness, all 9 possible ascendant genealogies (B; I–IX) possess a fixed set of exactly one each of the 6 paths shown in (C). Each child has exactly one female parent, one male parent, one maternal and one paternal grandfather, and one maternal and one paternal grandmother, even if e.g., their maternal grandfather and their paternal grandfather is the same individual (as in B I). In this way, all genealogies of any given diameter have a single invariant set of ascendant paths, independent of their actual shape. (D) This invariant set of paths can be represented as a directed acyclic graph in which each node represents a class of relative (e.g., maternal grandfather, paternal grandfather, etc.), rather than a distinct individual. An individual may occupy more than one node (as e.g., in B I, where the same individual is both maternal grandfather and paternal grandfather), but a node may not contain more than one individual in an ascendant lineage.\n\nA 3-degree ascendant, 4-degree descendent SIG is shown with ascendant (I), descendant (III) and lateral (II) kin path sets identified as described in Figure 1. SIGs were constructed for all focal individuals (represented by the black node) who have a complete set of documented third-degree ascendant relatives (I), in which all their known relatives are represented. For each focal individual, the numbers of local and global kin (local kin and global kin variables in Table 1) in each ascendant (I) and lateral (II) kin class were counted each month. We estimated how well the reproductive success of focal individuals (descendent relatives in path set III) was predicted by cohabitation of kin classes in path sets I and II. ϕ = degree of relatedness.\n\n(A) We generated 1000 seed populations that satisfied group size constraints for all zoos, with individuals randomly assigned to locations. (B) A local optimization algorithm was applied to each seed population. This algorithm generated “mutated” populations that randomly reassigned new locations to groups of 50 individuals from the seed population, rejecting populations that did not satisfy group size constraints, to generate five new populations. We forecast the overall reproductive success for these five “mutated” populations and the original population, and selected the global optimum of all six populations. (C) The globally optimal population was then used to start the next cycle of mutation-selection, and the total process repeated 40 times.\n\n\nReferences\n\nWildt DE, Ellis S, Janssen D, et al.: Toward more effective reproductive science for conservation. Reproductive Science and Integrated Conservation. 2003; 2–20. Publisher Full Text\n\nComizzoli P, Crosier AE, Songsasen N, et al.: Advances in reproductive science for wild carnivore conservation. Reprod Domest Anim. 2009; 44(Suppl 2): 47–52. PubMed Abstract | Publisher Full Text\n\nPope TR: Reproductive success increases with degree of kinship in cooperative coalitions of female red howler monkeys (Alouatta seniculus). Behav Ecol Sociobiol. 2000; 48(4): 253–267. Publisher Full Text\n\nKrakauer AH: Kin selection and cooperative courtship in wild turkeys. Nature. 2005; 434(7029): 69–72. PubMed Abstract | Publisher Full Text\n\nRussell AF, Hatchwell BJ: Experimental evidence for kin-biased helping in a cooperatively breeding vertebrate. Proc Biol Sci. 2001; 268(1481): 2169–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMalcolm JR, Marten K: Natural Selection and the Communal Rearing of Pups in African Wild Dogs (Lycaon pictus). Behav Ecol Sociobiol. 1982; 10(1): 1–13. Reference Source\n\nClutton-Brock TH, Brotherton PN, O’Riain MJ, et al.: Individual contributions to babysitting in a cooperative mongoose, Suricata suricatta. Proc Biol Sci. 2000; 267(1440): 301–305. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClutton-Brock TH, O’Riain MJ, Brotherton PN, et al.: Selfish sentinels in cooperative mammals. Science. 1999; 284(5420): 1640–1644. PubMed Abstract | Publisher Full Text\n\nGriffin AS, Pemberton JM, Brotherton PNM, et al.: A genetic analysis of breeding success in the cooperative meerkat (Suricata suricatta). Behav Ecol. 2003; 14(4): 472–480. Publisher Full Text\n\nClutton-Brock T: Cooperation between non-kin in animal societies. Nature. Nature Publishing Group; 2009; 462(7269): 51–7. PubMed Abstract | Publisher Full Text\n\nHamilton WD: The Genetical Evolution of Social Behaviour. I. J Theor Biol. 1964; 7(1): 1–16. PubMed Abstract | Publisher Full Text\n\nHamilton WD: The Genetical Evolution of Social Behaviour. II. J Theor Biol. 1964; 7(1): 17–52. PubMed Abstract | Publisher Full Text\n\nWright S: Coefficients of Inbreeding and Relationship. Am Nat. The University of Chicago Press for The American Society of Naturalists; 1922; 56(645): 330–338. Publisher Full Text\n\nBreiman L, Friedman JH, Olshen RA, et al.: Classification and Regression Trees. The Wadsworth statisticsprobability series. 1984. Reference Source\n\nBrowning LE, Patrick SC, Rollins LA, et al.: Kin selection, not group augmentation, predicts helping in an obligate cooperatively breeding bird. Proc Biol Sci. 2012; 279(1743): 3861–3869. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNowak MA: Evolving cooperation. J Theor Biol. 2012; 299: 1–8. PubMed Abstract | Publisher Full Text\n\nClutton-Brock J: A Natural History of Domesticated Mammals. 2nd ed. The Beagle: Records of The Museums And Art Galleries of The Northern Territory. Cambridge: The Cambridge University Press; 1999. Reference Source\n\nHardesty BD, Hubbell SP, Bermingham E: Genetic evidence of frequent long-distance recruitment in a vertebrate-dispersed tree. Ecol Lett. 2006; 9(5): 516–25. PubMed Abstract | Publisher Full Text\n\nSteinitz O, Troupin D, Vendramin GG, et al.: Genetic evidence for a Janzen-Connell recruitment pattern in reproductive offspring of Pinus halepensis trees. Mol Ecol. 2011; 20(19): 4152–64. PubMed Abstract | Publisher Full Text\n\nFile AL, Murphy GP, Dudley SA: Fitness consequences of plants growing with siblings: reconciling kin selection, niche partitioning and competitive ability. Proc Biol Sci. 2012; 279(1727): 209–218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKimble C: North American Meerkat Studbook. Silver Springs, MD, USA: Association of Zoos and Aquariums; 2012.\n\nEyre S: Australasian Meerkat Studbook. Sydney, Australia: Zoo and Aquarium Association Australasia; 2012.\n\nNewman MEJ: A measure of betweenness centrality based on random walks. Soc Networks. 2005; 27(1): 39–54. Publisher Full Text\n\nTherneau T, Atkinson E, Sinnwell J, et al.: kinship2: Pedigree functions. R package Version 1.3.7. [Internet]. 2012. Reference Source\n\nR Development Core Team: R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. R Foundation for Statistical Computing, Vienna, Austria. 2013. Reference Source\n\nDe’ath G, Fabricius KE: Classification and regression trees: A powerful yet simple technique for ecological data analysis. Ecology. 2000; 81(11): 3178–3192. Publisher Full Text\n\nElith J, Leathwick JR, Hastie T: Boosted regression trees - a new technique for modelling ecological data. J Anim Ecol. 2008; 77: 802–813. Publisher Full Text\n\nBureau A, Dupuis J, Falls K, et al.: Identifying SNPs predictive of phenotype using random forests. Genet Epidemiol. 2005; 28(2): 171–182. PubMed Abstract | Publisher Full Text\n\nKruskal WH, Wallis WA: Use of Ranks in One-Criterion Variance Analysis. J Am Stat Assoc. 1952; 47(260): 583–621. Publisher Full Text\n\nNewman S, Eyre S, Kimble C, et al.: Dataset 1 in: Reproductive success is predicted by social dynamics and kinship in managed animal populations. F1000Research. 1952. Data Source"
}
|
[
{
"id": "17367",
"date": "02 Nov 2016",
"name": "Tim Clutton-Brock",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAll fine – but the conclusions are incorporated in a way that makes them difficult for readers to assimilate. Both the Abstract and the Results need to explain how kin and group dynamics affect breeding success (i.e. which components of fitness are affected, in which direction and to what extent). The authors also need to be clearer about how and why they believe their results affect game and inclusive theory interactions; how they reconcile predictions based on these two approaches; and why they believe that the results of studies of captive animals of this kind provide insight into the evolutionary processes operating in natural populations.",
"responses": []
},
{
"id": "18031",
"date": "09 Dec 2016",
"name": "Alan H. Krakauer",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 strength of this manuscript lies in its analytical methods. Treatment of data, use of a network framework for highlighting the role of different types of relatives, and the statistical models, are generally well described. I found it impressive that the models performed as well as they did on the independent dataset (models developed using North American records were tested on data from the Australian populations). The approaches described here might be useful beyond inbred tightly-managed captive conservation settings, and could potentially help reveal the importance of multiple types of genealogical relationships in complex societies.\nTwo possible issues with appropriateness of the data: First, how parentage, and paternity in particular, is determined is not described in the text. Speaking with a zoo director about this, I learned that this is usually, but not always clear from behavior in the colony. Additionally, the text does not state how castrated individuals were treated (meerkat colony management occasionally involves neutering males to reduce aggression). The latter would obviously be extremely important for understanding direct reproductive success. The issue of whether human-sterilized individuals should be lumped together with “naturally” reproductively suppressed individuals is interesting in itself but not discussed in the manuscript. My guess is that neither of these issues change the data sufficiently to alter the outcome of the analyses, but it would be nice to know more.\nIt does not appear that the analysis took into account the identity of the zoo. It seems plausible to me that zoos could differ in the quality of care and housing in ways that are confounded with their decisions about meerkat group management. Since I’m not sure this paper makes specific management recommendations and seems to be more proof-of-concept of the analytical methods, I don’t necessarily see this as too damning.\nI don’t think the distinction between “game theory” and “kin selection theory” is necessarily accurate, nor particularly useful for the message of the paper. For example, see the following for a recent paper which uses a game theoretic approach to ask behavioral questions in an inclusive fitness context: Akçay &Van Cleve, 20161.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-870
|
https://f1000research.com/articles/5-305/v1
|
09 Mar 16
|
{
"type": "Data Note",
"title": "A curated transcriptome dataset collection to investigate the development and differentiation of the human placenta and its associated pathologies",
"authors": [
"Alexandra K. Marr",
"Sabri Boughorbel",
"Scott Presnell",
"Charlie Quinn",
"Damien Chaussabel",
"Tomoshige Kino",
"Sabri Boughorbel",
"Scott Presnell",
"Charlie Quinn",
"Damien Chaussabel",
"Tomoshige Kino"
],
"abstract": "Compendia of large-scale datasets made available in public repositories provide a precious opportunity to discover new biomedical phenomena and to fill gaps in our current knowledge. In order to foster novel insights it is necessary to ensure that these data are made readily accessible to research investigators in an interpretable format. Here we make a curated, public, collection of transcriptome datasets relevant to human placenta biology available for further analysis and interpretation via an interactive data browsing interface. We identified and retrieved a total of 24 datasets encompassing 759 transcriptome profiles associated with the development of the human placenta and associated pathologies from the NCBI Gene Expression Omnibus (GEO) and present them in a custom web-based application designed for interactive query and visualization of integrated large-scale datasets (http://placentalendocrinology.gxbsidra.org/dm3/landing.gsp). We also performed quality control checks using relevant biological markers. Multiple sample groupings and rank lists were subsequently created to facilitate data query and interpretation. Via this interface, users can create web-links to customized graphical views which may be inserted into manuscripts for further dissemination, or e-mailed to collaborators for discussion. The tool also enables users to browse a single gene across different projects, providing a mechanism for developing new perspectives on the role of a molecule of interest across multiple biological states. The dataset collection we created here is available at: http://placentalendocrinology.gxbsidra.org/dm3.",
"keywords": [
"Transcriptomics",
"Bioinformatics",
"Placenta",
"Trophoblast",
"Diabetes",
"Pre-eclampsia",
"IUGR",
"trophoblast differentiation"
],
"content": "Introduction\n\nWe aimed to make available via an interactive web-based application a collection of transcriptome datasets curated from the GEO public repository for their relevance to human placental development and pathology.\n\nThe placenta is a fetal organ indispensable for the establishment and maintenance of pregnancy. It connects the fetus to the maternal uterine wall via the umbilical cord, supplies nutrients and oxygen to the fetus, allows elimination of fetal waste, protects it from maternal infections and produces various hormones required for maintaining pregnancy1. The placental trophoblasts play critical roles for all of these activities by acting as an interface between fetus and mother2. In early embryogenesis, trophoblasts are the first cells differentiating from the fertilized egg, and eventually fusing with each other: a process transforming the monolayer cytotrophoblasts into syncytiotrophoblasts3. Several morphological and biochemical changes occur during this fusion process throughout pregnancy (known as trophoblast differentiation)4 to guarantee the development and appropriate functionality of the placenta1. Failure in placental formation, differentiation and function, particularly by trophoblast dysfunction, impacts fetal development and is associated with a wide range of pathologies, including gestational diabetes, hypertension, pre-eclampsia and intrauterine growth restriction (IUGR) of the fetus5. In addition, exposure of placenta to toxic compounds by a mother’s cigarette smoking alters the transcriptome of placental and fetal cells6. Indeed, maternal tobacco use causes various pregnancy-associated problems, including spontaneous fetal abortion, placental abruption, preterm birth, stillbirth, fetal growth restriction, and sudden infant death syndrome6,7.\n\nWith over 65,000 data deposited into the NCBI Gene Expression Omnibus (GEO), a public repository for functional genomics data, it can be challenging to identify datasets relevant to a particular research area. Indeed, GEO was primarily designed for the purpose of data archiving rather than data browsing and downstream analysis. Thus, we identified datasets from GEO particularly relevant to our interest in the development and pathology of the human placenta and uploaded them to the custom web-based Gene Expression Browser application (GXB) (http://placentalendocrinology.gxbsidra.org/dm3/landing.gsp), which provides seamless access to the data. GXB allows browsing and interactive visualization of large volumes of heterogeneous data. It also provides access to rich contextual information essential for data interpretation, such as: detailed gene information, relevant literature, study design, and sample information. In addition, the user can customize data plots by adding multiple layers of parameters, modify the sample order and generate links that can be shared via e-mail or used in future publications.\n\nThus we provide a resource enabling browsing of datasets relevant to placental development and pathology that offers a unique opportunity to identify genes that play a role in placental development/differentiation and are commonly modulated in pregnancy-associated diseases.\n\n\nMaterial and methods\n\n111 datasets, potentially relevant to the development and pathology of the human placenta, were identified in GEO using the following search query: “Homo sapiens[Organism] AND placenta[DESC]”. The majority of retrieved datasets were generated using Illumina or Affymetrix microarrays besides a few other commercial platforms.\n\nThe relevance of each one of the entries retreived with our query pertaining to “development and pathology of the human placenta” was assessed on a case-by-case basis. This process included reading the NCBI’s GEO-linked article and examining the list of samples profiled and their annotations as well as the study design. We selected for studies using primary placenta cells, placenta tissue or trophoblast cell lines comparing a) healthy with diseased samples or smoker vs. non-smoker or b) placenta cells in different differentiation/gestational stages. Finally we retrieved 24 curated datasets encompassing 759 transcriptome profiles, including 22 datasets generated using primary placenta cells and 2 datasets generated using trophoblast cell lines. Among the 24 datasets, there are 18 studies comparing control vs. diseased/smoker placenta cells, and 6 investigating transcriptomic changes during placental development and/or trophoblast differentiation. Among the many noteworthy datasets, several stood out, such as an extensive study comparing the diseased placentas with pre-eclampsia or unexplained IUGR vs. normal placentas (GSE24129)8. The datasets that comprise our collection are listed in Table 1.\n\nFor more information, see http://placentalendocrinology.gxbsidra.org/dm3/geneBrowser/list: PLAC1, placenta specific 1: CSH1, placental lactogen: XIST, X-inactive specific transcript NP: not published, no PubMed publication for this data set.\n\nOnce a final selection had been made, each dataset was downloaded from GEO in the SOFT file format. These files were then uploaded to the “placentaendocrinology” instance of GXB (http://placentalendocrinology.gxbsidra.org/dm3), an interactive web-based application developed at the Benaroya Research Institute, hosted on the Amazon Web Services cloud9. Information about samples and study design was also uploaded. Samples were then grouped based on relevant study variables and genes were ranked based on specified group comparisons. Details of the GXB software are described in a recent publication9. A tutorial for this software is also available online (https://gxb.benaroyaresearch.org/dm3/tutorials.gsp#gxbtut). GXB provides the user with a means to navigate and filter the dataset collection available at (http://placentalendocrinology.gxbsidra.org/dm3). Briefly, the datasets of interest can be quickly identified either by filtering with the pre-existing criteria listed in the column located in the left side of the dataset navigation window or by entering a query term in the search box located at the top of the window. Clicking one of the studies listed in the dataset navigation window opens a viewer, which is designed to support interactive browsing and graphic representations of the data in an interpretable format. This interface was developed to navigate ranked gene lists and to display expression results in a figure with a context-rich environment. Selecting a gene from the rank-ordered list located on the left side of the navigation window displays its expression values on the interactive plot. The drop-down menus located directly above the graphical display provide the user the following functions: a) Exporting the created graph as a portable network graphics (png) image or a csv file for performing a separate analysis. b) Changing ranking of genes in the list; this function allows the user to manipulate the ways of ranking the genes depending on his/her interest, or to include only the genes selected based on specific biological interest. c) Changing grouping of the samples (by using “Group Set” button); for example, the user can convert the groups created based on “cell type” to those on “disease type”. d) Identifying individual samples within a group using categorical or continuous variables (e.g., mode of delivery, ethnic group and age). e) Toggling between the bar chart view and a box plot view, with demonstration of the values as a single plot for each sample. Samples are split into different categories or groups whatever they are displayed in a bar chart or box plot. f) Providing a color legend for sample groups. g) Providing a color legend for the categorical information overlaid at the bottom of the graph. h) Selection of categorical information that is to be overlaid at the bottom of the graph. Using this function, the user can display, for example, gender or smoking status, at the bottom of the figure. The data without contextual information have no intrinsic utility. It is therefore important to capture and display the context information in the graph, so that the viewer can interpret the data shown in the graph. GXB provides functions for organizing the context information in the tabs located just above the graphical display. The tabs can be hidden to make more room for graphs, or be reappeared by clicking the blue “Show Info Panel” button located on the top right corner of the graphical display. Information on the genes selected from the list located in the left side of the graphical display is shown under the “Gene” tab. Information on the study for the demonstrated dataset is available under the “Study” tab. Detailed information of individual samples is provided under the “Sample” tab. Rolling the mouse cursor over a sample in either the bar chart or box plot view while displaying the “Sample” tab displays any clinical, demographic, or laboratory information available for that sample. Finally, the “Downloads” tab allows the advanced users to retrieve the original data for further analysis using other softwares/tools. It also provides all available sample annotation data along with the expression data. Other functionalities are provided under the “Tools” drop-down menu located in the top right corner of the user interface. Some of the notable functionalities available through this menu include: a) Annotations, which provides access to all the ancillary information about the study, samples and dataset organized across different tabs; b) Cross project view, which provides the ability to browse through all available studies while focusing on a the currently selected gene; c) Copy link, which generates a mini-URL encapsulating information about the display settings in use and that can be saved and shared with others (clicking on the envelope icon on the toolbar inserts the url in an email message via the local email client); d) Chart options, which gives user the option to customize chart labels.\n\n\nDataset validation\n\nWe performed quality control checks on the datasets loaded on GXB by validating the expression of placental marker genes. Specifically, we used placenta specific 1 (PLAC1)10 and human placental lactogen (CSH1)11 (Table 1). As expected, all samples from the datasets we incorporated in our collection expressed these two placental markers (hyperlinked for easy access in Table 1), except some samples in GSE12216, GSE28551 and GSE10588, where CSH1 was not measured.\n\nGender specific expression of the XIST transcript (in the samples for which sex information was available) was also examined to determine its concordance with demographic information provided with the GEO datasets (Table 1). An overall accordance of a high XIST expression in female samples compared to a low XIST expression in male samples was determined in our entire dataset collection (Table 1). Based on our experience, concordance should be close to 100%. Levels of concordance closer to 50%, which were not observed here, would indicate a potential error with handling of samples during processing that may dramatically affect data analysis and interpretation (e.g. plate inversion).\n\n\nData availability\n\nAll datasets included in our curated collection are available publically at the NCBI GEO website: http://www.ncbi.nlm.nih.gov/gds/. They were cited in our manuscript along with their GEO accession numbers (e.g. GSE24129). Signal files and sample description files for each uploaded GEO dataset can also be downloaded from the GXB tool under the “downloads” tab after accessing our dataset collection.",
"appendix": "Author contributions\n\n\n\nAM and TK conceived the theme for this dataset collection. AM, SB, SP and CQ contributed to the query, selection, loading and curation of datasets. DC and AM prepared the first draft of the manuscript. TK edited 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\nAll authors listed on this publication affiliated with the Sidra Medical and Research Center received support from the Qatar Foundation.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe would like to thank all the investigators who decided to make their datasets publicly available by depositing them into GEO.\n\n\nReferences\n\nBurton GJ, Fowden AL: The placenta: a multifaceted, transient organ. Philos Trans R Soc Lond B Biol Sci. 2015; 370(1663): 20140066. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSilva JF, Serakides R: Intrauterine trophoblast migration: A comparative view of humans and rodents. Cell Adh Migr. 2016; 1–23. PubMed Abstract | Publisher Full Text\n\nJi L, Brkic J, Liu M, et al.: Placental trophoblast cell differentiation: physiological regulation and pathological relevance to preeclampsia. Mol Aspects Med. 2013; 34(5): 981–1023. PubMed Abstract | Publisher Full Text\n\nSitras V, Fenton C, Paulssen R, et al.: Differences in gene expression between first and third trimester human placenta: a microarray study. PLoS One. 2012; 7(3): e33294. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang S, Regnault TR, Barker PL, et al.: Placental adaptations in growth restriction. Nutrients. 2015; 7(1): 360–89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVotavova H, Dostalova Merkerova M, Fejglova K, et al.: Transcriptome alterations in maternal and fetal cells induced by tobacco smoke. Placenta. 2011; 32(10): 763–70. PubMed Abstract | Publisher Full Text\n\nBruin JE, Gerstein HC, Holloway AC: Long-term consequences of fetal and neonatal nicotine exposure: a critical review. Toxicol Sci. 2010; 116(2): 364–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNishizawa H, Ota S, Suzuki M, et al.: Comparative gene expression profiling of placentas from patients with severe pre-eclampsia and unexplained fetal growth restriction. Reprod Biol Endocrinol. 2011; 9: 107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpeake C, Presnell S, Domico K, et al.: An interactive web application for the dissemination of human systems immunology data. J Transl Med. 2015; 13: 196. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCocchia M, Huber R, Pantano S, et al.: PLAC1, an Xq26 gene with placenta-specific expression. Genomics. 2000; 68(3): 305–12. PubMed Abstract | Publisher Full Text\n\nSamaan N, Yen SC, Friesen H, et al.: Serum placental lactogen levels during pregnancy and in trophoblastic disease. J Clin Endocrinol Metab. 1966; 26(12): 1303–8. PubMed Abstract | Publisher Full Text\n\nDunk CE, Roggensack AM, Cox B, et al.: A distinct microvascular endothelial gene expression profile in severe IUGR placentas. Placenta. 2012; 33(4): 285–93. PubMed Abstract | Publisher Full Text\n\nFounds SA, Conley YP, Lyons-Weiler JF, et al.: Altered global gene expression in first trimester placentas of women destined to develop preeclampsia. Placenta. 2009; 30(1): 15–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhou Y, Gormley MJ, Hunkapiller NM, et al.: Reversal of gene dysregulation in cultured cytotrophoblasts reveals possible causes of preeclampsia. J Clin Invest. 2013; 123(7): 2862–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVotavova H, Dostalova Merkerova M, Krejcik Z, et al.: Deregulation of gene expression induced by environmental tobacco smoke exposure in pregnancy. Nicotine Tob Res. 2012; 14(9): 1073–82. PubMed Abstract | Publisher Full Text\n\nGuo L, Tsai SQ, Hardison NE, et al.: Differentially expressed microRNAs and affected biological pathways revealed by modulated modularity clustering (MMC) analysis of human preeclamptic and IUGR placentas. Placenta. 2013; 34(7): 599–605. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHerse F, Dechend R, Harsem NK, et al.: Dysregulation of the circulating and tissue-based renin-angiotensin system in preeclampsia. Hypertension. 2007; 49(3): 604–11. PubMed Abstract | Publisher Full Text\n\nHuuskonen P, Storvik M, Reinisalo M, et al.: Microarray analysis of the global alterations in the gene expression in the placentas from cigarette-smoking mothers. Clin Pharmacol Ther. 2008; 83(4): 542–50. PubMed Abstract | Publisher Full Text\n\nSitras V, Paulssen R, Leirvik J, et al.: Placental gene expression profile in intrauterine growth restriction due to placental insufficiency. Reprod Sci. 2009; 16(7): 701–11. PubMed Abstract | Publisher Full Text\n\nMuehlenbachs A, Fried M, Lachowitzer J, et al.: Genome-wide expression analysis of placental malaria reveals features of lymphoid neogenesis during chronic infection. J Immunol. 2007; 179(1): 557–65. PubMed Abstract | Publisher Full Text\n\nHighet AR, Khoda SM, Buckberry S, et al.: Hypoxia induced HIF-1/HIF-2 activity alters trophoblast transcriptional regulation and promotes invasion. Eur J Cell Biol. 2015; 94(12): 589–602. PubMed Abstract | Publisher Full Text\n\nUusküla L, Männik J, Rull K, et al.: Mid-gestational gene expression profile in placenta and link to pregnancy complications. PLoS One. 2012; 7(11): e49248. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMikheev AM, Nabekura T, Kaddoumi A, et al.: Profiling gene expression in human placentae of different gestational ages: an OPRU Network and UW SCOR Study. Reprod Sci. 2008; 15(9): 866–77. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeng T, Chen H, Sun M, et al.: Identification of differential gene expression profiles in placentas from preeclamptic pregnancies versus normal pregnancies by DNA microarrays. OMICS. 2012; 16(6): 301–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWinn VD, Gormley M, Paquet AC, et al.: Severe preeclampsia-related changes in gene expression at the maternal-fetal interface include sialic acid-binding immunoglobulin-like lectin-6 and pappalysin-2. Endocrinology. 2009; 150(1): 452–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTsai S, Hardison NE, James AH, et al.: Transcriptional profiling of human placentas from pregnancies complicated by preeclampsia reveals disregulation of sialic acid acetylesterase and immune signalling pathways. Placenta. 2011; 32(2): 175–82. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "12824",
"date": "23 Mar 2016",
"name": "Brian Cox",
"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 web based utility is well assembled and easy to navigate and have very nice layout and graphic capabilities. A large spectrum of RNA microarray human placental sample sets have been assembled. Reservations are:The verification of the datasets using CSH1 and PLAC1 is rather minimal. Can you verify the differential expression observed in the originating manuscripts? At least in a couple of examples, such as one smoking and one preeclampsia/IUGR. Figures with examples graphics from these validations could be helpful. In many sample sets there exist batch effects. In some cases the depositors have identified the batches, but have not corrected for these, in other cases batches can be observed even when not formally identified by the depositor. For maximum utility these effects should be removed or a tool supplied to identify and remove them.Further to the need for batch correction, viewing a gene across multiple uncorrected/non-normalized data sets is not very informative.The web utility was frequently unavailable during the review process. A more stable platform or redundant server system should be established to limit the down time of this application. What user load can the server handle, how many concurrent users can the server handle?Only transcription arrays are used in the assembled data sets, there are numerous data sets involving DNA methylation, microRNAs and protein. Incorporation of these data sets would create a true systems level database of the human placenta.Improved utility would come from enabling merging of multiple datasets to perform meta analysis of aggregate analysis. This would require the installation of a batch corrections scheme or method/s. There is also a growing resource of sequencing based data sets that have been neglected in this application/manuscript. Once converted into a table of gene expression/counts, it should not be too challenging to incorporate these datasets as well. User training could be improved with additional figures to support descriptions of the utility and data sets. Better would be a vignette containing worked examples of the different types of analyses to generate the table and figures discussed in the paper. Similar worked vignette examples are frequently found in R libraries.",
"responses": [
{
"c_id": "1953",
"date": "11 May 2016",
"name": "Alexandra Marr",
"role": "Author Response",
"response": "The web based utility is well assembled and easy to navigate and have very nice layout and graphic capabilities. A large spectrum of RNA microarray human placental sample sets have been assembled. Reservations are:1. The verification of the datasets using CSH1 and PLAC1 is rather minimal. Can you verify the differential expression observed in the originating manuscripts? At least in a couple of examples, such as one smoking and one preeclampsia/IUGR. Figures with examples graphics from these validations could be helpful. We included a new figure (new Figure 1) showing the verification of differential gene expression in GXB compared to the values published in the originating manuscript. We also included the following paragraph in the “Dataset Validation” section of the manuscript: “We also verified and compared the differential expression using the Fold Change values obtained from GXB or the original manuscript. For this verification process, two datasets were selected GSE24129 and GSE30032. As shown in Figure 1, Fold Change values obtained from GXB correlate with those obtained from the original published article.”2. In many sample sets there exist batch effects. In some cases the depositors have identified the batches, but have not corrected for these, in other cases batches can be observed even when not formally identified by the depositor. For maximum utility these effects should be removed or a tool supplied to identify and remove them.Further to the need for batch correction, viewing a gene across multiple uncorrected/non-normalized data sets is not very informative.The reviewer raises an important point. Batch effects resulting in non-biological experimental variation across multiple batches of microarray experiments have been well documented. Several methods are available to correct for batch effects, such as Combat in ‘R’ (http://www.r-project.org/). In the context of a dataset compendium batch effects should be considered at two different levels: a) Within datasets: Unfortunately batch-processing information is available for only a small minority of datasets deposited in GEO (6% in the case of our “placental endocrinology” dataset collection), which makes systematically correcting for such effects impossible. This limitation of course affects any investigator inclined to use GEO as a resource. The practice of randomizing groups across runs, which avoids having batch effects confounding the analysis, is fortunately widely accepted. But it cannot be altogether discounted.However, this is where one of the distinct advantage of working with a compendium of dataset comes into play: a conclusion regarding differential expression of a given gene can be based on observation of such differences in not just one but multiple datasets, obtained independently by different investigators, in different parts of the world, using different array platforms. The conclusions that can be thus derived are especially robust. This point is illustrated by one of our recent contribution to F1000Research: http://f1000research.com/articles/4-89/v1b) Across datasets: We have previously implemented strategies for meta-analyses per-se, focusing on blood transcriptome data, and found it necessary to reduce the information from each dataset to p-values (comparison within each dataset of cases to a control group; 17724127, 16461797) or co-clustering information (module repertoire analyses 24662387). While GXB is primarily designed as a data browser it is possible to employ it similarly (but with added flexibility) for “meta-interpretation” (rather than meta-analysis). This consists in interpreting findings for a given gene across tens or hundreds of datasets, thus affording a unique perspective to the investigators and an effective means to identify and address knowledge gaps. This again is illustrated in the article mentioned above: http://f1000research.com/articles/4-89/v1. Notably, the cross-project view functionality also provides an independent search for datasets with genes that have a certain fold-changes above a chosen threshold. 3. The web utility was frequently unavailable during the review process. A more stable platform or redundant server system should be established to limit the down time of this application. What user load can the server handle, how many concurrent users can the server handle? We thank the reviewer for this critical feedback. Our instance was created on the Amazon Elastic Compute Cloud (Amazon Web services). The downtime was not due to the GXB application per se but rather to some missing packages related to the tomcat server. These packages have been fixed and the downtime issue is now resolved. The number of concurrent users is configurable and it is related to tomcat web server. The current configuration is 200 concurrent users. This can be reconfigured as needed.4. Only transcription arrays are used in the assembled data sets, there are numerous data sets involving DNA methylation, microRNAs and protein. Incorporation of these data sets would create a true systems level database of the human placenta. These are great ideas. We are currently working on the interactive web application (GXB) to be able to expand to other platforms such as working on SomaLogic data etc. We do see the establishment of our instance as an ongoing project, where additional datasets can be uploaded on demand (see “Request to load a Sample Set” function on the upper right corner of the web application page <http://placentalendocrinology.gxbsidra.org/dm3/geneBrowser/list>), for example when new datasets are published and deposited, and according to advances in our GXB technology. 5. Improved utility would come from enabling merging of multiple datasets to perform meta analysis of aggregate analysis. This would require the installation of a batch corrections scheme or method/s. We agree with the reviewer’s point of view. However, it is a difficult problem to address at the gene level. We are currently working on approaches and tools involving those modular repertoires (please see Chaussabel D. et al. 2008 Immunology 29:150-64 and Chaussabel D. & Baldwin N. 2014 Nat Rev Immunol. 14:271-80). See also our comments above pertaining to the point raised earlier regarding batch correction. 6. There is also a growing resource of sequencing based data sets that have been neglected in this application/manuscript. Once converted into a table of gene expression/counts, it should not be too challenging to incorporate these datasets as well. We agree that it would be very valuable to include RNAseq datasets in the future. We are not at this time in measure to accommodate this data type routinely but as a proof of principle a trial RNA-seq dataset have been setup to display RNA-seq profiles of immune cell subsets isolated from subjects with a wide range of diseases (GSE60424, https://gxb.benaroyaresearch.org/dm3/geneBrowser/show/396, http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109760).7. User training could be improved with additional figures to support descriptions of the utility and data sets. Better would be a vignette containing worked examples of the different types of analyses to generate the table and figures discussed in the paper. Similar worked vignette examples are frequently found in R libraries. Indeed, thank you for suggesting this. We started working on a ‘User Training’ document with detailed, step-by-step descriptions. This document will be published in the Collective Data Access channel of F1000Research very soon."
}
]
},
{
"id": "12946",
"date": "06 Apr 2016",
"name": "Maria Belen Rabaglino",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 of this article have collected several gene expression datasets, related with the development of the human placenta and associated pathologies, from the public database Gene Expression Omnibus (GEO) to analyze them in the context of a web application known as Gene Expression Browser (GXB) platform.This web tool provides a graphical interface to search and visualize gene status across different datasets. The collection of datasets presented on this article makes this platform particularly useful to gather information about transcriptome profiles related to human placenta and associated pathologies. Undoubtedly, this tool allows fast searching of a gene of interest on those datasets, which could facilitate visualization of results from the corresponding studies and helps in manuscripts or grants writing.There is no information on this manuscript about the statistical analysis followed to rank the genes on each dataset but it refers to the publication that details the GXB software1. Here, it is specified that genes are ranked according their differential expression using the limma package, and the links to the R scripts are showed. However, the application should include the information about the normalization/background correction methods used on each dataset. Different methods could yield different expression measures2 and so modify the gene ranking.Also, comparisons of gene expression across studies could be a powerful feature of this program, but for that is necessary to apply a method to remove batch effects.Another suggestion is to extend the verification of the datasets including genes that are known to be deregulated in placental pathologies --such as LEP up-regulation in preeclamptic placenta3,4--. An explanation about these genes along with graphs obtained from the web application could be useful for the reader as an example of its utility.Finally, the application should include the date of the last update. Obviously, new placenta-related datasets might be uploaded into the GEO database and so they should be added to the collection.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-305
|
https://f1000research.com/articles/5-270/v1
|
03 Mar 16
|
{
"type": "Research Note",
"title": "IL-6 stimulates a concentration-dependent increase in MCP-1 in immortalised human brain endothelial cells",
"authors": [
"Jai Min Choi",
"Odunayo O. Rotimi",
"Simon J. O'Carroll",
"Louise F.B. Nicholson",
"Jai Min Choi",
"Odunayo O. Rotimi",
"Simon J. O'Carroll"
],
"abstract": "Systemic inflammation is associated with neurodegeneration, with elevated interleukin-6 (IL-6) in particular being correlated with an increased risk of dementia. The brain endothelial cells of the blood brain barrier (BBB) serve as the interface between the systemic circulation and the brain microenvironment and are therefore likely to be a key player in the development of neuropathology associated with systemic inflammation. Endothelial cells are known to require soluble IL-6 receptor (sIL-6R) in order to respond to IL-6, but studies in rat models have shown that this is not the case for brain endothelial cells and studies conducted in human cells are limited. Here we report for the first time that the human cerebral microvascular cell line, hCMVEC, uses the classical mIL-6R signalling pathway in response to IL-6 in a concentration-dependent manner as measured by the production of monocyte chemotactic protein (MCP-1). This novel finding highlights a unique characteristic of human brain endothelial cells and that further investigation into the phenotype of this cell type is needed to elucidate the mechanisms of BBB pathology in inflammatory conditions.",
"keywords": [
"IL-6 stimulation",
"systemic inflammation",
"MCP-1",
"cell phenotype"
],
"content": "Abbreviations\n\nAD: Alzheimer’s disease; BBB: Blood brain barrier; BD: Becton Dickinson; BSA: Bovine serum albumin; CBA: Cytometric bead array: FACS: fluorescent activated cell sorting; FBS: Fetal bovine serum; gp130: Glycoprotein 130; hCMEC: Human cardiac microvascular endothelial cells; hCMVEC: Human cerebral microvascular endothelial cell; hFGF: human fibroblastic growth factor; HUVEC: Human umbilical vein endothelial cell; IL-6: Interleukin-6; IL-8: Interleukin-8; JAK: Janus kinase; M199: Mediium 199; MCP-1: Monocyte chemoattractant protein-1; mIL-6R: Membrane –bound interleukin-6 receptor; sIL-6r: Soluble interleukin-6 receptor; STAT: Signal transducers and activators of transcription; TNFα: Tumour necrosis factor alpha; VCAM-1: Vascular cell adhesion protein-1.\n\n\nIntroduction\n\nIL-6 is a pleiotropic cytokine that exerts it effects by binding to an IL-6 receptor, that exists in both a soluble form (sIL-6R) and a membrane-bound form (mIL-6R), that then associates with the membrane-bound glycoprotein 130 (gp130) receptor to initiate intracellular signalling pathways including the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway1. gp130 is expressed ubiquitously throughout the body whereas mIL-6R is only expressed in a few cell types including hepatocytes, monocytes, B cells and neutrophils. These cells are able to respond to circulating IL-6 in what is known as classical IL-6 signalling. Cells types such as endothelial cells that do not express mIL-6R are thus dependent on the trans signalling pathway, wherein neutrophils release sIL-6R2. sIL-6R binds to free IL-6 to form a sIL-6R/IL-6 complex which can then interact with gp130 to initiate cell signalling pathways. This has been demonstrated in studies using human umbilical vein endothelial cells (HUVEC) where IL-6 alone did not elicit a response, while the sIL-6R/IL-6 complex induced the production of IL-6, interleukin-8 (IL-8) and monocyte chemotactic protein (MCP-1)3. It appears that brain endothelial cells may however exhibit a different phenotype, with a response to IL-6 alone shown in primary cultures of rat brain endothelial cells4,5. Such phenotypic differences are of interest as brain endothelial cells not only form a major component of the blood brain barrier (BBB), but elevated levels of circulating serum IL-6 have been correlated with increased risk of developing dementia6. Furthermore, a large meta-analysis of 40 cross-sectional studies found Alzheimer’s disease patients had elevated serum levels of a number of pro-inflammatory cytokines including IL-6 when compared to healthy control subjects7. Since sIL-6 trans-signalling is thought to be pro-inflammatory while the classic mIL-6 signalling is mainly anti-inflammatory1, the nature of the response of brain endothelial cells to IL-6 is very important. We serendipitously found, in a related study, that human brain endothelial cells show a unique response to IL-6. Thus we explored the response of human cerebral microvascular endothelial cells (hCMVEC) following treatment with IL-6, by measuring the concentration of four pro-inflammatory cytokines (IL-6, Il-8, VCAM-1 and MCP-1), in order to better describe the phenotype of these human brain endothelial cells.\n\n\nMaterials and methods\n\nThe human cerebral microvascular endothelial cell (hCMVEC) line was purchased from Applied Biological Materials, Inc. The hCMVEC cell line was cultured in M199 media supplemented with 10% fetal bovine serum (HyClone), 1μg/mL hydrocortisone (Sigma), 3ng/mL hFGF (PeproTech), 10ng/mL hEGF (PeproTech), 10μg/mL heparin (Sigma), 1% GlutaMAX (Gibco), 1% Pen-Strep (Sigma), and 80μM dibutyryl-cAMP (Sigma). All cell culture surfaces were coated with collagen I (Gibco) at a concentration of 1μg/cm2.\n\nFor treatment, cells were plated in M199 medium supplemented with 2% fetal bovine serum, 110 nM hydrocortisone, 1% GlutaMAX, 1% Pen-Strep, and 80μM dibutyryl-cAMP for 3 days at which time the media was replaced with media supplemented with varying concentrations of IL-6 (PeproTech) (0.1ng/mL, 1ng/mL, 10ng/mL, and 100ng/mL). 100μL of media was collected at 72 hours post treatment from each well and centrifuged at 420 × G for 10 minutes to remove any cellular debris. 80μL of the supernatant was stored at -80°C until needed.\n\nSoluble IL-6, IL-8, VCAM-1 and MCP-1 were measured by multiplexed cytometric bead array (CBA; BD Biosciences, San Jose, CA, USA; see http://www.bdbiosciences.com/documents/CBA). These were selected as previous studies carried out in our laboratory8 show that they are highly inducible in the hCMVEC cells, whilst having different functions in the inflammatory response: proinflammatory cytokine (IL-6, IL-8), leukocyte adhesion (VCAM-1) and leukocyte recruitment (MCP-1). The CBA was conducted according to the manufacturer’s instructions (except 25 μl of conditioned media was used instead of 50 μl)9. For each cytokine measured, a 10-point standard curve (1–5000 pg/ml) was included. CBA samples were analysed using a BD Accuri C6 Flow Cytometer (BD Biosciences). Data were analysed using FCAP Array software (version 3.0.1, BD Biosciences) which automatically converts the sample mean fluorescent intensity values to pg/ml concentration based on the standard curve.\n\nA master cocktail containing the proteins of interest (as supplied by BD Biosciences) at a concentration of 5000pg/mL in CBA assay diluent was prepared. 10 standard solutions of the following concentrations were then prepared using the standard diluent (BD Biosciences): 5000pg/mL, 2500pg/mL, 1250pg/mL, 625pg/mL, 312.5pg/mL, 158.25pg/mL, 78.125pg/mL, 39.0625pg/mL, 19.53125pg/mL and 0pg/mL.\n\nThe capture beads supplied with the kit for the proteins of interest (CBA Flex set numbers; IL-6, 558276; IL-8, 558277; sVCAM-1, 560427; MCP-1, 558287) were vortexed for 15 seconds and diluted 1:100 in bead diluent to make up the bead cocktail. 25μL of the bead cocktail was added to 25μL of sample/standard in a 96-well plate. The plate was mixed on a shaker at 500rpm for 5 minutes before incubating for 1 hour at room temperature. The detector cocktail was prepared in the same way as the bead cocktail, substituting the capture beads for detector antibodies (as per the BD manufacturer’s protocol) and the bead diluent for detector diluent. 25μL of the detector cocktail was then added to the wells and the plate mixed as before and left to incubate at room temperature for 2 hours. After incubation was complete 200μL of wash buffer (BD Biosciences) was added to each well and the plate centrifuged at 1300rpm for 10 minutes at room temperature. The supernatant was discarded and the beads re-suspended in 50μL of wash buffer before being transferred into a tube for analysis.\n\nA BD Accuri C6 Flow Cytometer was used to measure the fluorescence of the samples/standards. Using the FCAP Array software version 3.0.1, BD Biosciences), the mean fluorescent intensities of the standards were used to generate a standard curve for each protein of interest. The mean fluorescent intensities of the samples were measured against the standard curve to give the concentrations of the proteins in pg/mL. This data was exported to Microsoft Excel 2010 (Microsoft Corporation) for processing.\n\nStudent’s t-test with two-tailed distribution and correction for two samples with unequal variances was carried out on data obtained from CBA using Microsoft Excel 2010. Results were deemed to be significant if the p-value was less than 0.05.\n\n\nResults\n\nhCMVEC cells were shown to be responsive to IL-6 treatment alone (Figure 1). A clear concentration dependent effect was observed in MCP-1 release, with IL-6 concentrations as low as 1ng/mL producing a statistically significant increase in MCP-1 (Figure 1D). Such a response was not found for release of IL-6, IL-8 and VCAM-1 (Figure 1A–C, respectively). Interestingly, no difference was found in IL-6 levels between the media control and treated wells. We also observed that the BSA vehicle caused a decrease in VCAM-1 levels similar to that seen with 100ng/mL IL-6 treatment (Figure 1C). Given that the scale of release is so small (approximately 70pg/mL) it is difficult to reach a meaningful conclusion about the production of VCAM-1.\n\nhCMVEC cells were treated with varying concentrations of IL-6 for 72 hours before the media was collected for CBA analysis. The media was assayed for markers of endothelial inflammation IL-6 (A), IL-8 (B), VCAM-1 (C) and MCP-1 (D). Each graph represents the mean ± SD (n=3). Statistical significance was evaluated using Student’s t-test by comparison to the media control; * p<0.05; ** p<0.01; *** p<0.001.\n\n\nDiscussion\n\nTo date, the effects of IL-6 on brain endothelial cells have been studied mostly in cells derived from rats, in which IL-6 was found to stimulate eicosanoid production, increase permeability, and increase trans-cellular transport of the human immunodeficiency virus4,5,10. This would suggest that rat brain endothelial cells are able to utilise the classical pathway of IL-6 signalling by binding directly to the membrane form of the receptor, mIL-6R. Additionally, in vivo studies in rats have shown that deletion of mIL-6R on brain endothelial cells attenuated the development of fever, further supporting the involvement of mIL-6R in pathological processes11. However, such findings in animal models do not always translate into humans. Differences in the signalling pathways of IL-6 between humans and rats have been demonstrated in astrocytes. Human astrocytes were found to require the presence of sIL-6R in order to respond to IL-6 whereas rat astrocytes were able to response to IL-6 alone12. It is not yet known if similar differences exist between human and rat brain endothelial cells. There are few studies examining the response of human brain endothelial cells to IL-6, mostly focusing on permeability13,14. Nor are there any available studies investigating the immune-modulatory effects of IL-6 in human brain endothelial cells. We have conclusively shown chemokine production in response to IL-6 alone in a human-derived brain endothelial cell line. We found that IL-6 treatment alone resulted in a concentration-dependent release of MCP-1 in hCMVEC cells but did not induce production of IL-6, IL-8 or VCAM-1. The fact that hCMVEC cells respond to IL-6 in the absence of the addition of sIL-6R suggests that hCMVEC cells may be expressing mIL-6R. These preliminary results open the unexpected possibility that human brain endothelial cells may be able to utilise both the classical (mIL-6R-mediated) and trans (sIL-6R-mediated) pathways of IL-6 signalling through the presence of mIL-6R and the possible proteolytic cleavage or differential mRNA splicing to form sIL-6R. Furthermore, the lack of IL-6 and IL-8 induction in the hCMVEC response to IL-6 compared to the HUVEC response to the sIL-6/IL-6 complex3 suggests that signalling through mIL-6R and sIL-6R elicits different responses. A possible explanation for this can be found in hepatocytes, where the expression of gp130 is much higher than mIL-6R so that only a small proportion of gp130 receptors will be activated with IL-6 stimulation, whereas the sIL-6R/IL-6 complex can activate all gp130 receptors on the cell resulting in a much stronger effect1. Our finding that hCMVEC cells show a concentration-dependent increase in MCP-1 in response to IL-6 is important, as MCP-1 has been shown to be involved in increasing BBB permeability in an in vitro co-culture model of rat brain endothelial cells and astrocytes15. The production of MCP-1 by brain endothelial cells helps to explain the effects of IL-6 on BBB permeability.\n\nIt is worth noting that the IL-6 concentration was similar between IL-6 treatment groups and control groups. One might expect that the higher concentration treatment groups would have a higher concentration of IL-6 since a greater amount of IL-6 was added. It would be interesting to know if the added IL-6 was internalised by the cells via receptor-mediated endocytosis, as is known to occur in hepatocytes16. While IL-6 is rapidly internalised, the sIL-6R/IL-6 complex undergoes very little internalisation prolonging its signalling capabilities, and adding to the differences between sIL-6R-mediated signalling and mIL-6R-mediated signalling1.\n\nIn BB19 cells, another immortalised brain endothelial cell line, VCAM-1 production is reported to be increased 2-fold compared to baseline after 8 hours of IL-6 stimulation. This then declines 24 hours post treatment17. While it is possible that we simply did not observe an increase in VCAM-1 production after IL-6 stimulation it had declined back to baseline by our 72 hour time point, our 72 hour time point was chosen specifically as an earlier study from our laboratory8 has shown that hCMVEC cells produce substantial amounts of many inflammatory markers at 72 hours post treatment with TNF α in comparison to 24 and 48 hours post-treatment. We also found that our BSA vehicle caused a significant decrease in VCAM-1. The amount of BSA in the vehicle treatment group is equivalent to that of the 100ng/mL IL-6 treatment group raising the possibility that the decreased VCAM-1 production with 100ng/mL IL-6 treatment might be due to the BSA. Although there is a study documenting the inhibitory effect of BSA on TNFα-induced endothelial VCAM-1 production18, these authors used much higher concentrations of BSA and only measured the decrease in relation to the induced levels of VCAM-1, not basal levels. Despite the short comings of our study, the findings are significant and pave the way for future studies to further develop this research. For example, HUVEC could be included as a control, specific receptor blocking antibodies used to test the specificity of IL-6 supplementation and repeating this work using microvascular cells of a different origin (for example, human cardiac microvascular endothelial cells; hCMEC) would expand this study and thus provide further evidence supporting the proposal of a unique human brain endothelial cell phenotype.\n\n\nConclusion\n\nThere is a paucity of studies investigating the effects of IL-6 in human brain endothelial cells. Our study is the first to report findings suggesting that the human cerebral microvascular cell line, hCMVEC, responds to IL-6 in a way that it is unlikely to be through the soluble sIL-6R but instead uses the classical mIL-6R signalling pathway in a concentration-dependent manner as measured by the production of monocyte chemotactic protein (MCP-1). This is in contrast to previous studies in HUVEC cells that do not respond to IL-6 in the absence of sIL-6R. Thus there is no doubt that hCMVEC cells show a different immunoreactive phenotype to other endothelial cells. This highlights the heterogeneity in immunoreactive phenotypes among endothelial cell subtypes. The response of human brain endothelial cells to pro-inflammatory molecules should be further explored to elucidate the mechanisms of BBB pathology in inflammatory diseases such an Alzheimer’s disease.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for effect of IL-6 on hCMVEC expression of inflammatory markers (Figure 1), 10.5256/f1000research.8153.d11498519",
"appendix": "Author contributions\n\n\n\nLN conceived the study. SO designed the experiments. OR and JC carried out the research. JC wrote the first draft of the manuscript. SO and LN contributed to the experimental design and all authors we involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported through a Grant from the Maurice and Phyllis Paykel Trust; Project ID 3702741.\n\n\nAcknowledgements\n\nNo additional acknowledgements.\n\n\nReferences\n\nRose-John S: IL-6 trans-signaling via the soluble IL-6 receptor: importance for the pro-inflammatory activities of IL-6. Int J Biol Sci. 2012; 8(9): 1237–1247. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarnes TC, Anderson ME, Moots RJ: The many faces of interleukin-6: the role of IL-6 in inflammation, vasculopathy, and fibrosis in systemic sclerosis. Int J Rheumatol. 2011; 2011: 721608. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRomano M, Sironi M, Toniatti C, et al.: Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment. Immunity. 1997; 6(3): 315–325. PubMed Abstract | Publisher Full Text\n\nde Vries HE, Hoogendoorn KH, van Dijk J, et al.: Eicosanoid production by rat cerebral endothelial cells: Stimulation by lipopolysaccharide, interleukin-1 and interleukin-6. J Neuroimmunol. 1995; 59(1–2): 1–8. PubMed Abstract | Publisher Full Text\n\nde Vries HE, Blom-Roosemalen MC, van Oosten M, et al.: The influence of cytokines on the integrity of the blood-brain barrier in vitro. J Neuroimmunol. 1996; 64(1): 37–43. PubMed Abstract | Publisher Full Text\n\nKoyama A, O'Brien J, Weuve J, et al.: The role of peripheral inflammatory markers in dementia and alzheimer's disease: A meta-analysis. J Gerontol A Biol Sci Med Sci. 2013; 68(4): 433–440. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSwardfager W, Lanctot K, Rothenburg L, et al.: A meta-analysis of cytokines in Alzheimer’s disease. Biol Psychiatry. 2010; 68(10): 930–941. PubMed Abstract | Publisher Full Text\n\nO'Carroll SJ, Kho DT, Wiltshire R, et al.: Pro-inflammatory TNFα and IL-1β differentially regulate the inflammatory phenotype of brain microvascular endothelial cells. J Neuroinflammation. 2015; 12: 131. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBurkert K, Moodley K, Angel CE, et al.: Detailed analysis of inflammatory and neuromodulatory cytokine secretion from human NT2 astrocytes using multiplex bead array. Neurochem Int. 2012; 60(6): 573–580. PubMed Abstract | Publisher Full Text\n\nDohgu S, Fleegal-DeMotta MA, Banks WA: Lipopolysaccharide-enhanced transcellular transport of HIV-1 across the blood-brain barrier is mediated by luminal microvessel IL-6 and GM-CSF. J Neuroinflammation. 2011; 8: 167. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEskilsson A, Mirrasekhian E, Dufour S, et al.: Immune-induced fever is mediated by IL-6 receptors on brain endothelial cells coupled to STAT3-dependent induction of brain endothelial prostaglandin synthesis. J Neurosci. 2014; 34(48): 15957–15961. PubMed Abstract | Publisher Full Text\n\nOh JW, Van Wagoner NJ, Rose-John S, et al.: Role of IL-6 and the soluble IL-6 receptor in inhibition of VCAM-1 gene expression. J Immunol. 1998; 161(9): 4992–4999. PubMed Abstract\n\nDuchini A, Govindarajan S, Santucci M, et al.: Effects of tumor necrosis factor-alpha and interleukin-6 on fluid-phase permeability and ammonia diffusion in CNS-derived endothelial cells. J Investig Med. 1996; 44(8): 474–482. PubMed Abstract\n\nRochfort KD, Collins LE, Murphy RP, et al.: Downregulation of blood-brain barrier phenotype by proinflammatory cytokines involves NADPH oxidase-dependent ROS generation: Consequences for interendothelial adherens and tight junctions. PLoS One. 2014; 9(7): e101815. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStamatovic SM, Shakui P, Keep RF, et al.: Monocyte chemoattractant protein-1 regulation of blood-brain barrier permeability. J Cereb Blood Flow Metab. 2005; 25(5): 593–606. PubMed Abstract | Publisher Full Text\n\nWang Y, Fuller GM: Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes. Mol Biol Cell. 1994; 5(7): 819–828. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrudhomme JG, Sherman IW, Land KM, et al.: Studies of Plasmodium falciparum cytoadherence using immortalized human brain capillary endothelial cells. Int J Parasitol. 1996; 26(6): 647–655. PubMed Abstract | Publisher Full Text\n\nZhang WJ, Frei B: Albumin selectively inhibits TNF alpha-induced expression of vascular cell adhesion molecule-1 in human aortic endothelial cells. Cardiovasc Res. 2002; 55(4): 820–829. PubMed Abstract | Publisher Full Text\n\nChoi JM, Rotimi OO, O'Carroll SJ, et al.: Dataset 1 in: IL-6 stimulates a concentration-dependent increase in MCP-1 in immortalised human brain endothelial cells. F1000Research. 2016. Data Source"
}
|
[
{
"id": "12723",
"date": "11 Mar 2016",
"name": "Robert B Petersen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 preliminary study on the effect of IL6 on an immortalized human cerebral microvascular cell line. The authors show convincingly that addition of IL6, in the absence of exogenous sIL6R, induces production of MCP-1, a chemotactic protein. This induction is a subset of possible responses shown previously in response to IL1-ß and TNF-alpha by the same group. The result also contrasts with an earlier report on the response of HUVEC cells in which the authors showed that the response required sIL6R (the cell culture conditions were quite different than the present study).The report would be stronger if the authors had showed either the presence of mIL6R on the cell surface or demonstrated the absence of sIL6R in the system.",
"responses": [
{
"c_id": "1867",
"date": "16 Mar 2016",
"name": "Louise Nicholson",
"role": "Author Response",
"response": "Thank you for your very positive and constructive feedback. We shall definitely include a sentence or two in our manuscript acknowledging the benefits of future work to confirm the presence of mIL-6R on the surface of our hCMVEC or the absence of sIL-6R in the system we have used."
}
]
},
{
"id": "13376",
"date": "14 Apr 2016",
"name": "Diego Gomez-Nicola",
"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 reported observations are of interest, to understand immune to brain communication. Given the background of the experiments and the potential interest, I have two suggestions of further experimental work that would provide strong support to the authors claims:As acknowledged by the authors, if IL6 alone can elicit a response, this would indicate the potential expression of mIL-6R by hCMVECs. This needs to be confirmed, as its needed to understand the already reported data. The authors comment on the different inflammatory profile caused by activation from mIL-6R or sIL6R. Seems like the current findings just scratch on the surface of that response, and further analysis of pro- vs anti-inflammatory mediators seems a sensible complement to this study.",
"responses": [
{
"c_id": "1932",
"date": "11 May 2016",
"name": "Louise Nicholson",
"role": "Author Response",
"response": "We thank the reviewer for the constructive feedback and are pleased that our observations add to an understanding of the immune to brain communication. We accept that these preliminary findings need to be explored further and agree that the two suggested experiments are very valuable indeed in confirming the expression of mIL-6R and the inflammatory profile resulting from the activation of mIL-6R or sIL-6R. We shall definitely include these recommendations in the discussion of our revised manuscript as a way of advancing these preliminary findings. We selected the category of a ’Research note’ for publication of our observations because they are preliminary and do indeed ‘just scratch on the surface’. We believe however, that the observations are important and of value to the field paving the way for future studies as suggested by the reviewer. At this stage we are not in a position to undertake any further experiments ourselves but would be delighted if someone else picked up the challenge."
}
]
}
] | 1
|
https://f1000research.com/articles/5-270
|
https://f1000research.com/articles/4-1422/v1
|
10 Dec 15
|
{
"type": "Research Article",
"title": "Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers",
"authors": [
"Kevin McKernan",
"Jessica Spangler",
"Lei Zhang",
"Vasisht Tadigotla",
"Yvonne Helbert",
"Theodore Foss",
"Douglas R. Smith",
"Jessica Spangler",
"Lei Zhang",
"Vasisht Tadigotla",
"Yvonne Helbert",
"Theodore Foss",
"Douglas R. Smith"
],
"abstract": "The Center for Disease Control estimates 128,000 people in the U.S. are hospitalized annually due to food borne illnesses. This has created a demand for food safety testing targeting the detection of pathogenic mold and bacteria on agricultural products. This risk extends to medical Cannabis and is of particular concern with inhaled, vaporized and even concentrated Cannabis products. As a result, third party microbial testing has become a regulatory requirement in the medical and recreational Cannabis markets, yet knowledge of the Cannabis microbiome is limited. Here we describe the first next generation sequencing survey of the microbial communities found in dispensary based Cannabis flowers and demonstrate the limitations in the culture-based regulations that are being superimposed from the food industry.",
"keywords": [
"Cannabis",
"Microbiome",
"Mycotoxins",
"Cannabidiol",
"Paxilline",
"Citrinin",
"qPCR",
"Culture",
"Next generation sequencing"
],
"content": "Introduction\n\nMany states in the U.S. are crafting regulations for microbial detection on Cannabis in absence of any comprehensive survey of Cannabis microbiomes. A few of these regulations are inducing growers to “heat kill” or pasteurize Cannabis flowers to lower microbial content. While this is a harmless suggestion, we must remain aware of how these drying techniques often create false negatives in culture-based safety tests used to monitor colony-forming units (CFU). Even though pasteurization may be effective at sterilizing some of the microbial content, it does not eliminate various pathogenic toxins or spores. Aspergillus spores and mycotoxins are known to resist pasteurization1,2. Similar thermal resistance has been reported for E. coli produced Shiga toxin3. While pasteurization may reduce CFU’s used in petri-dish or plating based safety tests, it does not reduce the microbial toxins, spores or DNA encoding these toxins.\n\nMycotoxin monitoring in Cannabis preparations is important since aflatoxin produced by Aspergillus species is a carcinogen. The clearance of aflatoxin requires the human liver enzyme CYP3A4 and this liver enzyme is potently inhibited by cannabinoids4,5. Modern day Cannabis flowers can produce up to 25% (w/v) cannabinoids presenting potent inhibition of CYP3A4 and CYP2C19. Health compromised patients exposed to aflatoxin and clearance-inhibiting cannabinoids raise new questions in regards to the current safety tolerances to aflatoxin. Similarly, Fusarium species are known to produce fungal toxins and has proven to be difficult to selectively culture with tailored media6–8. This is a common fault of culture-based systems as carbon sources are not exclusive to certain microbes and only 1% of microbial species are believed to be culturable9.\n\nWhile these risks have been well studied in the food markets, the presence of the microbial populations present on Cannabis flowers has never been surveyed with next generation sequencing techniques10–15. With the publication of the Cannabis genome16,17 and many other pathogenic microbial genomes, quantitative PCR assays have been developed that can accurately quantify fungal DNA present in Cannabis samples18. Here, we analyze the yeast and mold species present in 10 real world, dispensary-derived Cannabis samples by quantitative PCR and sequencing, and demonstrate the presence of several mycotoxin producing fungal strains that are not detected by widely used culture-based assays.\n\n\nMethods\n\n3.55ml of tryptic soy broth (TSB) was used to wet 250mg of homogenized flower in a whirlpack bag. TSB was aspirated from the reverse side of the 100μm mesh filter and placed into a BiolumixTM growth vial and spread onto a 3M Petri FilmTM and a SimPlateTM (3M PetrifilmTM 3M Microbiology, St. Paul, MN, USA; SimPlatesTM Biocontrol Systems, Bellevue, WA, USA; BioLumixTM Neogen, Lansing MI, USA) according to the respective manufacturers’ recommendations. BiolumixTM vials were grown and monitored for 48 hours while Petri-filmsTM and SimPlatesTM were grown for 5 days. Petri-filmsTM and SimPlatesTM were colony counted manually by three independent observers. Samples were tested on total coliform, total entero, total aerobic, and total yeast and mold. Only total yeast and mold discrepancies were graduated to sequencing.\n\nPlant DNA was extracted with SenSATIVAx according to manufacturers’ instructions (Medicinal Genomics part #420001). DNA was eluted with 50μl ddH20.\n\nPCR was performed using 5μl of DNA (3ng/μl) 12.5μl 2X LongAmp (NEB) with 1.25μl of each 10μM MGC-ITS3 and MGC-ITS3 primer (MGC-ITS3; TACACGACGTTGTAAAACGACGCATCGATGAAGAACGCAGC) and (MGC-ITS3R; AGGATAACAATTTCACACAGGATTTGAGCTCTTGCCGCTTCA) with 10μl ddH20 for a 25μl total reaction. An initial 95°C 5 minute denaturization was performed followed by 40 cycles of 95°C for 15s and 65°C for 90s. Samples were purified with 75μl SenSATIVAx, washed twice with 100μl 70% EtOH and bench dried for 5 minutes at room temperature. Samples were eluted in 25μl ddH20.\n\nDNA libraries were constructed with 250ng DNA using NEB’s NEBNext Quick ligation module (NEB # E6056S). End repair used 3μl of enzyme mix, 6.5μl of reagent mix, 55.5μl of DNA + ddH20. Reaction was incubated at 30°C for 20 minutes. After end repair, ligation was performed directly with 15μl of blunt end TA mix, 2.5μl of Illumina adaptor (10μM) and 1μl of ligation enhancer (assumed to be 20% PEG 6000). After 15 minute ligation at 25°C, 3μl of USER enzyme was added to digest the hairpin adaptors and prepare for PCR. The USER enzyme was tip-mixed and incubated at 37°C for 20 minutes. After USER digestion, 86.5μl of SenSATIVAx was added and mixed. The samples were placed on a magnet for 15 minutes until the beads cleared and the supernatant could be removed. Beads were washed twice with 150μl of 70% EtOH. Beads were left for 10 minute to air dry and then eluted in 25μl of 10mM Tris-HCl.\n\n25μl 2X Q5 polymerase was added to 23μl of DNA with 1μl of i7 index primer (25μM) and 1μl universal primer (25μm). After an initial 95°C for 10s, the library was amplified for 15 cycles of 95°C 10s, 65°C 90s. Samples were purified by mixing 75μl of SenSATIVAx into the PCR reaction. The samples were placed on a magnet for 15 minutes until the beads cleared and the supernatant could be removed. Beads were washed twice with 150μl of 70% EtOH. Beads were left for 10 minute to air dry and then eluted in 25μl of 10mM Tris-HCl. Samples were prepared for sequencing on the MiSeq version 2 chemistry according to the manufacturers’ instructions. 2×250bp reads were selected to obtain maximal ITS sequence information.\n\nPrimers described by Shirazi-zand et al. were utilized to amplify a segment of the 725bp PaxP gene. 25μl LongAmp (NEB) 4μl 10μM primer, 1μl DNA (14ng/μl), 20μl ddH20 to make a 50μl PCR reaction. Cycling conditions were slightly modified to accommodate a different polymerase. 95°C for 30s followed by 28 cycles of 95°C 15s, 55°C for 30s, 65°C 2.5 minutes. Samples were purified with 50μl of SenSATIVAx as described above. 1μl of purified PCR product was sized on Agilent HS 2000 chip. Nextera libraries and sequencing were performed according to instructions from Illumina using 2×75bp sequencing on a version 2 MiSeq.\n\nCitrinum forward GATTTTCCAAAATGCCGTCT and Citrinum reverse GCTCAAGCATTAATCTAGCTA primers were used with identical PCR conditions as above with the exception using 35 cycles of PCR. Samples were purified with 50μl of SenSATIVAx as described above. 1μl of purified PCR product was sized on Agilent HS 2000 chip. Nextera libraries and sequencing were performed according to instructions from Illumina using 2×75bp sequencing on a version 2 MiSeq. Reads were mapped to Genbank accession number LKUP01000000. Mappings were confirmed using BLAST to NCBI to ensure the strongest hits were to P. citrinum.\n\nReads were demultiplexed and trimmed with Casava 1.8.2 and trim_galore v0.4.1 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). FLASH v1.2.1119 was used to merge the reads using max_overlap 150. The reads were aligned to microbial references using MG-RAST v3.220. Alignments and classifications were confirmed with a second software tool from One Codex (https://onecodex.com/) and critical pathways identified for further evaluation with PCR of toxin producing genes. Reads are deposited in NCBI under SRA accession: SRP065410. Nextera 2×75bp sequencing of the PaxP gene was mapped to accession number HM171111.1 with CLCbio Workstation V4 at 98% identity over 80% of the read. One Codex analysis was put into Public mode under the following public URLs:\n\nAustralian Bastard:\n\nhttps://app.onecodex.com/analysis/public/201e7f1642e04a3c\n\nhttps://app.onecodex.com/analysis/public/58f1e03c10434bfa\n\nKD4:\n\nhttps://app.onecodex.com/analysis/public/2e86e262817246c4\n\nhttps://app.onecodex.com/analysis/public/1abd5b60446140a0\n\nKD6:\n\nhttps://app.onecodex.com/analysis/public/a92d3dff5485499d\n\nhttps://app.onecodex.com/analysis/public/8d72e2514e564ecd\n\nKD8:\n\nhttps://app.onecodex.com/analysis/public/8d72e2514e564ecd\n\nhttps://app.onecodex.com/analysis/public/d6e2e0bcfba3469f\n\nLiberty Haze:\n\nhttps://app.onecodex.com/analysis/public/7bcd650fa5544f2c\n\nhttps://app.onecodex.com/analysis/public/7f0feb6cb0a94d56\n\nGirls Scout Cookie:\n\nhttps://app.onecodex.com/analysis/public/a71b1ce8331c461d\n\nhttps://app.onecodex.com/analysis/public/8d6f10c7ee684f93\n\nJakes Grape:\n\nhttps://app.onecodex.com/analysis/public/bc8af5ed19e5407a\n\nhttps://app.onecodex.com/analysis/public/99d7a4a2f7af486b\n\nRECON:\n\nhttps://app.onecodex.com/analysis/public/8a22a16cc2e24731\n\nhttps://app.onecodex.com/analysis/public/0af6ae26a01f48d5\n\nGreenCrack:\n\nhttps://app.onecodex.com/analysis/public/6114843d2eb3425e\n\nhttps://app.onecodex.com/analysis/public/3eee642786c54a88\n\nLA Confidential:\n\nhttps://app.onecodex.com/analysis/public/01e8aefb0d4f4f62\n\nhttps://app.onecodex.com/analysis/public/b74c2988fcd84e38\n\nNYC Diesel:\n\nhttps://app.onecodex.com/analysis/public/441cfad759f64dcc\n\nhttps://app.onecodex.com/analysis/public/d97b39cae96c4a44\n\n\nResults\n\nA commercially available total yeast and mold qPCR assay (TYM-PathogINDICAtor, Medicinal Genomics, Woburn MA) was used to screen for fungal DNA in a background of host Cannabis DNA. The TYM qPCR assay targets the ribosomal DNA Internal Transcribed Spacer region 2 (ITS2) using modified primers described previously21,22. Fungal DNA amplified using these primers may also be subjected to next generation sequencing to identify the contributing yeast and mold species. ITS sequencing has been widely used to identify and enumerate fungal species present in a given sample23.\n\nWe purified DNA from Cannabis samples obtained from two different geographic regions (Amsterdam and Massachusetts) several years apart (2011 and 2015). The majority of samples purified and screened with ITS qPCR were negative for amplification signal implying reagents clean of fungal contamination. Six of the 17 dispensary-derived Cannabis samples tested positive for yeast and mold in the TYM qPCR assay. These results were compared with the results derived from three commercially available culture-based detection systems for each of the 17 samples (3M PetrifilmTM 3M Microbiology, St. Paul, MN, USA; SimPlatesTM Biocontrol Systems, Bellevue, WA, USA; BioLumixTM Neogen, Lansing MI, USA; Figure 1). Of the 6 qPCR positive samples, two tested negative in all 3 culture-based assays and four tested negative in 1 or 2 of the culture-based assays (Table 1). None of the qPCR negative samples tested positive in any of the culture-based assays. Each of the 6 discordant samples was subjected to ITS sequencing to precisely identify the collection of microbes present. Four additional samples from a different geographic origin (Amsterdam) were also subjected to ITS sequencing, for a total of 10 Cannabis samples.\n\nFigure 1A. qPCR signal from TYM (red line) test run concurrently (multiplexed) with a plant internal control marker (green line). This marker targets a conserved region in the Cannabis genome and should show up in every assay (upper left). SimPlates count the number of discolored wells (purple to pink) as a proxy for CFU/gram. Only total aerobic show growth (upper right). Petrifilm only demonstrate colonies on total aerobic platings (lower left). Biolumix demonstrate no signal across all 4 tests (lower right). Figure 1B. Sample KD8 fails to culture any total yeast and mold yet demonstrates significant TYM qPCR signal. Sample was graduated to ITS based next generation sequencing. Figure 1C. Sample Liberty Haze was tested with 3 culture based methods and compared to qPCR. Sample was graduated to ITS based next generation sequencing.\n\nBiolumix had the lowest sensitivity failing to pick up 4/17 samples detected with other culture-based platforms. qPCR identified 2 samples that were not picked up by any other method. Positive qPCR samples were sequenced to identify the contributing signal. Highlighted samples fail the 10,000 CFU/g cutoffs which equates to a Cq of 26 on the qPCR assay according to the manufacturers’ instructions. (f) is fail or over 10,000 CFU/g. (p) is pass or under 10,000 CFU/g. The raw CFU numbers can be deduced by dividing the CFU number by the 1,000 fold dilution factor used in this study.\n\nTNTC = Too Numerous To Count\n\nEach discordant sample presented with an array of microbial species, as shown in Figure 2. No sample presented with a single dominant species, and each sample displayed multiple species of interest. Of particular concern were the identified DNA sequences from toxin producing species: Aspergillus versicolor24–28, Aspergillus terreus29, Penicillium citrinum30–32, Penicillium paxilli33,34.\n\nPenicillium and Aspergillus are commonly found (Y axis) but at different read counts in each sample (X axis). Read counts are more a reflection of sample normalization for sequencing than inter sample quantitation provided by qPCR.\n\nWe further analyzed the ITS sequence alignments using the whole genome shotgun based microbiome classification software known as One Codex35. Nine of the ten samples sequenced showed the presence of P. paxilli (Figure 3). To verify the accuracy of this ITS phylotyping, a gene involved in the paxilline toxin biosynthesis pathway of P. paxilli was amplified with PaxPss1 and PaxPss2 primers described by Saikia et al.36 The resulting 725bp amplicon (expected size) was sequenced to confirm the presence of the P. paxilli biosynthesis gene in the Cannabis sample KD8 (Figure 4). This was successfully repeated with primers designed to target genes in the citrinin pathway of P. citrinum. There were some discrepancies between the results derived from the two software platforms (One Codex and MG-RAST). The MG-RAST analysis, using merged, paired reads correlated better with the PCR results. While One Codex predicted and confirmed KD8 as having the highest P. paxilli content, the One Codex platform is optimized for whole genome shotgun data and may not be able to differentiate the 18S sequence differences (391/412 aligned bases) between these two species with a K-mer based approach.\n\nP. paxilli is the most frequently found contaminant in Cannabis flowers. P. citrinum is not in the One Codex database at this time. One Codex utilizes a fast k-mer based approach for whole genome shotgun classification and can be influenced by read trimming and database content. The reads provided to MG-RAST were trimmed and FLASH’d (paired end reads merged when overlapping) prior to classification. K-mer based approaches can significantly differ from longer word size methods and this underscores the importance of confirmatory PCR in microbiome analysis.\n\nCitrinum primers we designed from Genbank accession number LKUP01000753. Paxilline primers were used as described in Saikia et al. PCR products were made into shotgun libraries with Nextera and sequenced on an Illumina MiSeq with 2×75bp reads to over 10,000X coverage. Reads were mapped with CLCbio 4 to NCBI accession number HM171111.1 (A) and LKUP01000000 respectively (B). Paired reads are displayed as blue lines, green and red lines are unpaired reads. Read coverage over the amplicons are depicted in a blue histogram over the cluster while paired end read distance is measured in a red histogram over the region. Off target read mapping is limited. P. paxilli mappings are displayed on top (A) and P. citrinum mappings are displayed on bottom (B). Alignment of PCR primers to P. paxilli reference shows a 5 prime mismatch that is a result of the primers being designed to target spliced RNA according to Saikia et al.\n\nWith the confirmed presence of P. paxilli, we are curious to find out whether the toxin, paxilline, is present in the samples. Development of monoclonal antibodies to paxilline has recently been described37, but commercial ELISA assays with sensitivity under 50ppb do not appear to be available at this time. A >50ppb multiplexed ELISA assay is available from Randox Food Diagnostics (Crumlin, UK). Detection with LC-MS/MS has also been described38,39, however, and experiments are underway to determine whether paxilline can be identified in the background of cannabinoids and terpenes present in Cannabis samples.\n\n\nDiscussion\n\nSeveral potentially harmful fungal species were detected in dispensary-derived Cannabis samples by qPCR and subsequent sequencing in this study. Three different culture-based assays failed to detect all of the positive samples and one, BioLumixTM, detected only one out of 7 positive samples. A review of the literature suggests that Penicillium microbes can be cultured on CYA media, but some may require colder temperatures (21-24C) and 7 day growth times40. Of the Penicillium, only P. citrinum has been previously reported to culture with 3M Petri-Film41. In addition, several studies have demonstrated plant phytochemicals and terpenoids like eugenol can inhibit the growth of fungi42. It is possible the different water activity of the culture assay compared to the natural terpene rich flower environment is contributing to the false negative test results.\n\nQuantitative PCR is agnostic to water activity and can be performed in hours instead of days. The specificity and sensitivity provides important information on samples that present risks invisible to culture based systems. The draw back to qPCR is the method’s indifference to living or non-living DNA. While techniques exist to perform live-dead qPCR, the live status of the microbes is unrelated to toxin potentially produced while the microbes were alive. ELISA assays exist to screen for some toxins43. Current state-recommended ELISA’s do not detect citrinin or paxilline, the toxins produced by P. citrinum and P. paxilli, respectively. The predominance of these Penicillium species in a majority of the samples tested is interesting. Several Penicillium species are known to be endophytes on various plant species, including P. citrinum10, and this raises the question of whether they are also Cannabis endophytes.\n\nPaxilline is a tremorgenic and ataxic potassium channel blocker and has been shown to attenuate the anti-seizure properties of cannabidiol in certain mouse models44–46. Paxilline is reported to have tremorgenic effects at nanomolar concentrations and is responsible for Ryegrass-staggers disease47. Cannabidiol is often used at micromolar concentrations for seizure reduction implying sub-percentage contamination of paxilline could still be a concern. Citrinin is a mycotoxin that disrupts Ca2+ efflux in the mitochondrial permeability transition pore (mPTP)48–55. Ryan et al. demonstrated that cannabidiol affects this pathway suggesting a potential concern for CBD-mycotoxin interaction56. Considering the hydrophobicity of paxilline and the recent interest in the use of cannabidiol derived from Cannabis flower oils for drug resistant epilepsy, more precise molecular screening of fungal toxins may be warranted57–62.\n\nOur survey of Cannabis flowers in this study was limited. Further screening will be required to define a set of tests that can adequately capture all risks. While ELISA assays are easy point of use tests that can be used to detect fungal toxins, they can suffer from lack of sensitivity and cross reactivity. ITS amplification and sequencing offers hypothesis-free testing that can complement the lack of specificity in ELISA assays. Appropriate primer design can survey a broad spectrum of microbial genomes while affording rapid iteration of design. Quantitative PCR has also demonstrated single molecule sensitivity and linear dynamic range over 5 orders of magnitude offering a very robust approach for detection of microbial risks. This may be important for the detection of nanomolar potency mycotoxins. Further studies are required to validate better detection methods for these toxins and verify whether paxilline or citrinin are present on Cannabis at concentrations that present a clinical risk.\n\n\nConclusions\n\nThese results demonstrate that culture based techniques superimposed from the food industry should be re-evaluated based on the known microbiome of actual Cannabis flowers in circulation at dispensaries. Several mycotoxin producing molds were detected that can potentially interfere with the medical use of cannabidiol. These microbes failed to grow on traditional culture-based platforms but were rapidly detected with molecular based techniques. Further studies are required to quantitate the presence and concentration of mycotoxin production.",
"appendix": "Author contributions\n\n\n\nKJM designed the study and performed the One-Codex analysis and PCR verifications.\n\nJS designed and ran the culture and qPCR laboratory experiments.\n\nLZ assisted in the figure generation and laboratory experiment.\n\nYH assisted in sequencing and PCR confirmation of Pax.\n\nVT- read alignment, MG-RAST, primer design and analysis.\n\nTF- Sample tracking software, figure generations, ITS software comparisons.\n\nDS- Manuscript construction and review.\n\n\nCompeting interests\n\n\n\nThe authors are employees of Medicinal Genomics Corporation (MGC). MGC manufactures qPCR reagents utilized in this study.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nJohn McPartland, Cindy Orser, Brad Douglass, Joost Heeroma, Nick Greenfield, Rebecca McKernan and Kellie Dodd for thoughtful advice.\n\n\nReferences\n\nFujikawa H, Itoh T: Tailing of thermal inactivation curve of Aspergillus niger spores. Appl Environ Microbiol. 1996; 62(10): 3745–3749. PubMed Abstract | Free Full Text\n\nKabak B, Dobson AD: Biological strategies to counteract the effects of mycotoxins. J Food Prot. 2009; 72(9): 2006–2016. PubMed Abstract\n\nRasooly R, Do PM: Shiga toxin Stx2 is heat-stable and not inactivated by pasteurization. Int J Food Microbiol. 2010; 136(3): 290–294. PubMed Abstract | Publisher Full Text\n\nLangouet S, Coles B, Morel F, et al.: Metabolism of aflatoxin B1 by human hepatocytes in primary culture. Adv Exp Med Biol. 1996; 387: 439–442. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nKocic-Tanackov S, Dimić G, Lević J, et al.: Effects of onion (Allium cepa L.) and garlic (Allium sativum L.) essential oils on the Aspergillus versicolor growth and sterigmatocystin production. J Food Sci. 2012; 77(5): M278–284. PubMed Abstract | Publisher Full Text\n\nSong F, Ren B, Chen C, et al.: Three new sterigmatocystin analogues from marine-derived fungus Aspergillus versicolor MF359. Appl Microbiol Biotechnol. 2014; 98(8): 3753–3758. PubMed Abstract | Publisher Full Text\n\nEl-Sayed Abdalla A, Zeinab Kheiralla M, Sahab A, et al.: Aspergillus terreus and its toxic metabolites as a food contaminant in some Egyptian Bakery products and grains. Mycotoxin Res. 1998; 14(2): 83–91. PubMed Abstract | Publisher Full Text\n\nAmes DD, Wyatt RD, Marks HL, et al.: Effect of citrinin, a mycotoxin produced by Penicillium citrinum, on laying hens and young broiler chicks. Poult Sci. 1976; 55(4): 1294–1301. PubMed Abstract | Publisher Full Text\n\nMazumder PM, Mazumder R, Mazumder A, et al.: Antimicrobial activity of the mycotoxin citrinin obtained from the fungus Penicillium citrinum. Anc Sci Life. 2002; 21(3): 191–197. PubMed Abstract | Free Full Text\n\nPark SY, Kim R, Ryu CM, et al.: Citrinin, a mycotoxin from Penicillium citrinum, plays a role in inducing motility of Paenibacillus polymyxa. FEMS Microbiol Ecol. 2008; 65(2): 229–237. PubMed Abstract | Publisher Full Text\n\nItoh Y, Johnson R, Scott B: Integrative transformation of the mycotoxin-producing fungus, Penicillium paxilli. Curr Genet. 1994; 25(6): 508–513. PubMed Abstract | Publisher Full Text\n\nShibayama M, Ooi K, Johnson R, et al.: Suppression of tandem-multimer formation during genetic transformation of the mycotoxin-producing fungus Penicillium paxilli by disrupting an orthologue of Aspergillus nidulans uvsC. Curr Genet. 2002; 42(1): 59–65. PubMed Abstract | Publisher Full Text\n\nMinot SS, Krumm N, Greenfield NB: One Codex: A Sensitive and Accurate Data Platform for Genomic Microbial Identification. bioRxiv. 2015. Publisher Full Text\n\nSaikia S, Parker EJ, Koulman A, et al.: Defining paxilline biosynthesis in Penicillium paxilli: functional characterization of two cytochrome P450 monooxygenases. J Biol Chem. 2007; 282(23): 16829–16837. PubMed Abstract | Publisher Full Text\n\nMaragos CM: Development and Evaluation of Monoclonal Antibodies for Paxilline. Toxins (Basel). 2015; 7(10): 3903–3915. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVishwanath V, Sulyok M, Labuda R, et al.: Simultaneous determination of 186 fungal and bacterial metabolites in indoor matrices by liquid chromatography/tandem mass spectrometry. Anal Bioanal Chem. 2009; 395(5): 1355–1372. PubMed Abstract | Publisher Full Text\n\nUhlig S, Egge-Jacobsen W, Vralstad T, et al.: Indole-diterpenoid profiles of Claviceps paspali and Claviceps purpurea from high-resolution Fourier transform Orbitrap mass spectrometry. Rapid Commun Mass Spectrom. 2014; 28(14): 1621–1634. PubMed Abstract | Publisher Full Text\n\nHoubraken J, Frisvad JC, Samson RA: Taxonomy of Penicillium section Citrina. Stud Mycol. 2011; 70(1): 53–138. PubMed Abstract | Publisher Full Text | Free Full Text\n\n3M. Reference Source\n\nZare M, Shams-Ghahfarokhi M, Ranjbar-Bahadori S, et al.: Comparative study of the major Iranian cereal cultivars and some selected spices in relation to support Aspergillus parasiticus growth and aflatoxin production. Iran Biomed J. 2008; 12(4): 229–236. PubMed Abstract\n\nLabs R: 2015. Reference Source\n\nShirazi-zand Z, Ahmad-Molaei L, Motamedi F, et al.: The role of potassium BK channels in anticonvulsant effect of cannabidiol in pentylenetetrazole and maximal electroshock models of seizure in mice. Epilepsy Behav. 2013; 28(1): 1–7. PubMed Abstract | Publisher Full Text\n\nSabater-Vilar M, Nijmeijer S, Fink-Gremmels J: Genotoxicity assessment of five tremorgenic mycotoxins (fumitremorgen B, paxilline, penitrem A, verruculogen, and verrucosidin) produced by molds isolated from fermented meats. J Food Prot. 2003; 66(11): 2123–2129. PubMed Abstract\n\nSánchez-Pastor, E, Andrade F, Sánchez-Pastor JM, et al.: Cannabinoid receptor type 1 activation by arachidonylcyclopropylamide in rat aortic rings causes vasorelaxation involving calcium-activated potassium channel subunit alpha-1 and calcium channel, voltage-dependent, L type, alpha 1C subunit. Eur J Pharmacol. 2014; 729: 100–106. PubMed Abstract | Publisher Full Text\n\nImlach WL, Finch SC, Dunlop J, et al.: The molecular mechanism of \"ryegrass staggers,\" a neurological disorder of K+ channels. J Pharmacol Exp Ther. 2008; 327(3): 657–664. PubMed Abstract | Publisher Full Text\n\nChagas GM, Campello AP, Kluppel ML: Mechanism of citrinin-induced dysfunction of mitochondria. I. Effects on respiration, enzyme activities and membrane potential of renal cortical mitochondria. J Appl Toxicol. 1992; 12(2): 123–129. PubMed Abstract | Publisher Full Text\n\nChagas GM, Oliveira BM, Campello AP, et al.: Mechanism of citrinin-induced dysfunction of mitochondria. II. Effect on respiration, enzyme activities, and membrane potential of liver mitochondria. Cell Biochem Funct. 1992; 10(3): 209–216. PubMed Abstract | Publisher Full Text\n\nChagas GM, Oliveira MA, Campello AP, et al.: Mechanism of citrinin-induced dysfunction of mitochondria. IV--Effect on Ca2+ transport. Cell Biochem Funct. 1995; 13(1): 53–59. PubMed Abstract | Publisher Full Text\n\nChagas GM, Oliveira MB, Campello AP, et al.: Mechanism of citrinin-induced dysfunction of mitochondria. III. Effects on renal cortical and liver mitochondrial swelling. J Appl Toxicol. 1995; 15(2): 91–95. PubMed Abstract | Publisher Full Text\n\nDa Lozzo EJ, Oliveira MB, Carnieri EG: Citrinin-induced mitochondrial permeability transition. J Biochem Mol Toxicol. 1998; 12(5): 291–297. PubMed Abstract | Publisher Full Text\n\nJeswal P: Citrinin-induced chromosomal abnormalities in the bone marrow cells of Mus musculus. Cytobios. 1996; 86(344): 29–33. PubMed Abstract\n\nRibeiro SM, Campello AP, Chagas GM, et al.: Mechanism of citrinin-induced dysfunction of mitochondria. VI. Effect on iron-induced lipid peroxidation of rat liver mitochondria and microsomes. Cell Biochem Funct. 1998; 16(1): 15–20. PubMed Abstract | Publisher Full Text\n\nRibeiro SM, Chagas GM, Campello AP, et al.: Mechanism of citrinin-induced dysfunction of mitochondria. V. Effect on the homeostasis of the reactive oxygen species. Cell Biochem Funct. 1997; 15(3): 203–209. PubMed Abstract | Publisher Full Text\n\nRyan D, Drysdale AJ, Lafourcade C, et al.: Cannabidiol targets mitochondria to regulate intracellular Ca2+ levels. J Neurosci. 2009; 29(7): 2053–2063. PubMed Abstract | Publisher Full Text\n\nDevinsky O, Whalley BJ, Di Marzo V: Erratum to: Cannabinoids in the Treatment of Neurological Disorders. Neurotherapeutics. 2015; 12(4): 910. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDevinsky O, Whalley BJ, Di Marzo V: Cannabinoids in the Treatment of Neurological Disorders. Neurotherapeutics. 2015; 12(4): 689–691. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFriedman D, Devinsky O: Cannabinoids in the Treatment of Epilepsy. N Engl J Med. 2015; 373(11): 1048–1058. PubMed Abstract | Publisher Full Text\n\nRosenberg EC, Tsien RW, Whalley BJ, et al.: Cannabinoids and Epilepsy. Neurotherapeutics. 2015; 12(4): 747–768. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCilio MR, Thiele EA, Devinsky O: The case for assessing cannabidiol in epilepsy. Epilepsia. 2014; 55(6): 787–790. PubMed Abstract | Publisher Full Text\n\nDevinsky O, Cilio MR, Cross H, et al.: Cannabidiol: pharmacology and potential therapeutic role in epilepsy and other neuropsychiatric disorders. Epilepsia. 2014; 55(6): 791–802. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11516",
"date": "17 Dec 2015",
"name": "John McPartland",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 succinct, well-written study has two major aims: 1. Utilize NextGen and qPCR to identify microorganisms inhabiting dispensary-sourced Cannabis flowers. 2. Compare results from these sequencing techniques to results from traditional culture-based methods. Results from the qPCR survey led to a third aim: confirm the presence of two heretofore unreported mycotoxin-producing fungi on Cannabis: Penicillium citrinum and Penicillium paxilli. Five critiques:1. “Microbiome” appears in the manuscript’s title, so you should cite some literature regarding plant microbiomes in general, as well as Cannabis-specific research. Vorholt (2012) and Turner et al. (2013) provide general overviews. Cannabis-specific microbiome studies (Kusari et al. 2013, Gautam et al. 2013) generated very different results than yours, and should be discussed. The rhizosphere study by Winston et al. (2014) ought to be mentioned, and highlight the complimentary nature of rhizosphere and phyllosphere studies.\n\nThe foliar microbiome (aka, phyllosphere, as opposed to rhizosphere) can be partitioned into two groups: epiphytes live upon the leaf epidermis, and endophytes occupy intercellular spaces within the leaf. Culture-based detection systems normally surface-sterilize plant samples, so they assume that cultured organisms are endophytes. NextGen and qPCR should identify both epiphytes and endophytes. Classic epiphytes identified in your study include Kabatiella (Aureobasidium) microsticta and Sarocladium (Acremonium) strictum.\n\nPhyllosphere organisms may be plant pathogens, and cause disease symptoms; diseased plants should never reach a dispensary. However, phyllosphere organisms may act as symbionts (good for the plant) or commensals (indifferent), and their asymptomatic presence is not easily detected. Nevertheless these cryptic organisms may cause disease in humans. The spores from phyllosphere fungi readily pass through waterpipes (Moody et al. 1982), and survive in smoke drawn from cannabis cigarettes (Kurup et al. 1983), as do aflatoxins (Llewellyn and O'Rear 1977). Worth mentioning. 2. Explain the methodology used to select three culture-based detection systems in this study. Are they the most widely-used systems? Are they the systems recommended by cannabis regulatory agencies? If the method was simply “convenience sampling,” say so. 3. Methods used in the qPCR ITS assay should be described in the Methods section, not the Results section. 4. Figure 2, “DNA sequencing of ITS2 amplicons from culture negative samples that are qPCR positive for total yeast and mold tests,” deserves some comment. Some of the taxa are not yeasts or molds. They include angiosperms (Zea mays, Pachysandra procumbens), a protozoan (Sterkiella histriomuscorum), and an “uncultured bacterium.” Comment please. 5. This study revealed a surprisingly depauperate Cannabis foliar microbiome, compared to a recent study of Genlisea species, using similar methods, that identified 92 genera of organisms (Cao et al. 2015). See Delmotte et al. (2009) for rich microbiomes in other plant species. Gzebenyuk (1984) isolated 79 species of fungi from hemp stems in Russia. Comment please.\n\nWhere are the bacteria? Much of the concern over microbiology and food safety focuses on human enteric pathogens (e.g., Escherichia coli, Salmonella spp.) and opportunistic bacteria (e.g., Pseudomonas aeruginosa, Berkholderia cepacia). Previous culture-based studies have isolated these organisms from Cannabis (e.g., Taylor et al. 1982, Ungerleider et al. 1982). The only bacterium you identify (in Figure 2, erroneously identified as a yeast or mold) is Pantoea agglomerans, formerly known as Enterobacter agglomerans, a gram-negative bacterium and an opportunistic human pathogen. Minor critiques:The detection and confirmation of Penicillium citrinum and Penicillium paxilli deserves mention in the abstract of the paper! An exhaustive review of the Cannabis literature (McPartland et al. 2000) found no references to these organisms.Species names should never be capitalized. For example, P. Citrinum should read P. citrinum. The full name should be spelled out the first time it appears in the Methods section, Penicillium citrinum.The Methods section should explain that the PaxP gene comes from Penicillium paxilli. The Methods section should identify NEP as New England BioLabs.Recommend some future directions: a comparison of indoor crops and outdoor crops (outdoor crops may show a seasonal community succession), and survey the microbiome of police seizures from well-provenanced foreign sources, such as Mexico.",
"responses": [
{
"c_id": "1871",
"date": "10 May 2016",
"name": "Kevin McKernan",
"role": "Author Response",
"response": "This succinct, well-written study has two major aims: 1. Utilize NextGen and qPCR to identify microorganisms inhabiting dispensary-sourced Cannabis flowers. 2. Compare results from these sequencing techniques to results from traditional culture-based methods. Results from the qPCR survey led to a third aim: confirm the presence of two heretofore unreported mycotoxin-producing fungi on Cannabis: Penicillium citrinum andPenicillium paxilli. Five critiques:1. “Microbiome” appears in the manuscript’s title, so you should cite some literature regarding plant microbiomes in general, as well as Cannabis-specific research. Vorholt (2012) and Turner et al. (2013) provide general overviews.Cannabis-specific microbiome studies (Kusari et al. 2013, Gautam et al. 2013) generated very different results than yours, and should be discussed. The rhizosphere study by Winston et al. (2014) ought to be mentioned, and highlight the complimentary nature of rhizosphere and phyllosphere studies. The foliar microbiome (aka, phyllosphere, as opposed to rhizosphere) can be partitioned into two groups: epiphyteslive upon the leaf epidermis, and endophytes occupy intercellular spaces within the leaf. Culture-based detection systems normally surface-sterilize plant samples, so they assume that cultured organisms are endophytes. NextGen and qPCR should identify both epiphytes and endophytes. Classic epiphytes identified in your study include Kabatiella (Aureobasidium) microsticta and Sarocladium (Acremonium) strictum. Phyllosphere organisms may be plant pathogens, and cause disease symptoms; diseased plants should never reach a dispensary. However, phyllosphere organisms may act as symbionts (good for the plant) or commensals (indifferent), and their asymptomatic presence is not easily detected. Nevertheless these cryptic organisms may cause disease in humans. The spores from phyllosphere fungi readily pass through waterpipes (Moody et al. 1982), and survive in smoke drawn from cannabis cigarettes (Kurup et al. 1983), as do aflatoxins (Llewellyn and O'Rear 1977). Worth mentioning. Author response: Several of the references cited relate to analysis of the bacterial microbiome on diverse plant species, which falls well beyond the scope of this study (focusing on the fungal microbiome of Cannabis). However, we added a brief mention of the very limited literature relating to Cannabis microbiomes to the introduction as follows:\"Our knowledge of the natural microbiome of field-grown Cannabis in terms of rhizosphere bacteria, and endophytic fungi is limited to just a few focused studies1-3. Very little is known about the potential for bacterial and fungal contamination on medicinal Cannabis. Nevertheless, many states in the U.S. are now crafting regulations for detection of microbial contamination on Cannabis in the absence of any comprehensive survey of actual samples. A few of these regulations are inducing growers to “heat kill” or pasteurize Cannabis flowers to lower microbial content. While this seems a harmless suggestion, we must remain aware of how these drying techniques may create false negatives in culture-based safety tests used to monitor colony-forming units (CFU). Even though pasteurization may be effective at sterilizing some of the microbial content, it does not eliminate various pathogenic toxins or spores. Aspergillus spores and mycotoxins are known to resist pasteurization 1, 2 . Similar thermal resistance has been reported for E. coli produced Shiga toxin 3 . While pasteurization may reduce CFU’s used in petri-dish or plating based safety tests, it does not reduce the microbial toxins, spores or DNA encoding these toxins.\"Author response: We also added a sentence to the end of the second paragraph of the discussion in reference to the two existing publications on Cannabis endophytic fungi. \"Several Penicillium species are known to be endophytes on various plant species, including P. citrinum 10 , and this raises the question of whether they are also Cannabis endophytes. Indeed, P. citrinum and a species identified as P. copticola (a member of the citrinun clade12) have previously been identified as Cannabis endophytes, along with several Aspergillus species2, 3.\" 2. Explain the methodology used to select three culture-based detection systems in this study. Are they the most widely-used systems? Are they the systems recommended by cannabis regulatory agencies? If the method was simply “convenience sampling,” say so. Author response: A sentence was added to the culture based methods section. The culture-based methods selected for testing here represent those currently in use by established medicinal Cannabis safety testing laboratories.3. Methods used in the qPCR ITS assay should be described in the Methods section, not the Results section. Author response: Paragraph 1 of the results section was moved to the methods section.4. Figure 2, “DNA sequencing of ITS2 amplicons from culture negative samples that are qPCR positive for total yeast and mold tests,” deserves some comment. Some of the taxa are not yeasts or molds. They include angiosperms (Zea mays, Pachysandra procumbens), a protozoan (Sterkiella histriomuscorum), and an “uncultured bacterium.” Comment please. Author response: The MG-RAST database contains multiple taxa. The hits to non-fungal species contained multiple mismatches and were deemed spurious. We filtered the data to remove all non-fungal hits and regenerated the figures, reporting all fungal species detected with 10 reads or more. This resulted in a higher number of species reported for some samples and fewer for others. Figure 2 was revised and the figure legend expanded. 5. This study revealed a surprisingly depauperate Cannabis foliar microbiome, compared to a recent study of Genliseaspecies, using similar methods, that identified 92 genera of organisms (Cao et al. 2015). See Delmotte et al. (2009) for rich microbiomes in other plant species. Gzebenyuk (1984) isolated 79 species of fungi from hemp stems in Russia. Comment please. Where are the bacteria? Much of the concern over microbiology and food safety focuses on human enteric pathogens (e.g., Escherichia coli, Salmonella spp.) and opportunistic bacteria (e.g., Pseudomonas aeruginosa, Berkholderia cepacia). Previous culture-based studies have isolated these organisms from Cannabis (e.g., Taylor et al. 1982, Ungerleider et al. 1982). The only bacterium you identify (in Figure 2, erroneously identified as a yeast or mold) isPantoea agglomerans, formerly known as Enterobacter agglomerans, a gram-negative bacterium and an opportunistic human pathogen.Author response: This study focuses only on the fungal microbiome. The studies cited are looking at field-grown samples. We revised the MG-RAST figures to include all fungal species detected down to a level of 10 reads. This reveals a significantly larger number of species in two samples. Other species display only a handful of species. Medicinal Cannabis is often grown indoors in artificial media. The first paragraph of the discussion section was expanded and split into two paragraphs as follows:\"This study demonstrates detection of numerous fungal species by molecular screening of ITS2 in several dispensary-derived Cannabis samples. These included the toxigenic Penicillium species: P. paxilli, P. citrinum, P. commune, P. chrysogenum, P. corylophilum, Aspergillus species: A. terreus, A. niger, A. flavus, A. versicolor and Eurotium repens. In addition, a pathogenic species Cryptococcus liquefaciens was detected. The fungal microbiomes of the different samples differed significantly in the number and diversity of species present. Two samples contained a large diversity of species, similar to previous studies that used field-grown samples and culture-based outgrowth methods2, 3, 11. Other samples contained only a few species at significant levels. This is perhaps not surprising given the prevalence of indoor culture methods using artificial growth media for medicinal Cannabis. However, we do not have any knowledge of the specific growth conditions that were used for the samples analyzed.Three different culture-based assays failed to detect all of the positive samples and one, BioLumix TM, detected only one out of 7 positive samples. A review of the literature suggests that Penicillium microbes can be cultured on CYA media, but some may require colder temperatures (21-24C) and 7 day growth times 40 . Of the Penicillium, only P. citrinum has been previously reported to culture with 3M Petri-Film 41 . It is possible the different water activity of the culture assay compared to the natural flower environment is contributing to the false negative test results.\" Minor critiques:1. The detection and confirmation of Penicillium citrinum and Penicillium paxilli deserves mention in the abstract of the paper! An exhaustive review of the Cannabis literature (McPartland et al. 2000) found no references to these organisms.Here we describe the first next generation sequencing survey of the fungal communities found in dispensary based Cannabis flowers by ITS2 sequencing, and demonstrate the sensitive detection of several toxigenic Penicillium and Aspergillus species, including P. citrinum and P. paxilli, that were not detected by one or more culture-based methods currently in use for safety testing.2. Species names should never be capitalized. For example, P. Citrinum should read P. citrinum. fixed3. The full name should be spelled out the first time it appears in the Methods section, Penicillium citrinum.fixed4. The Methods section should explain that the PaxP gene comes from Penicillium paxilli. The Methods section should identify NEP as New England BioLabs.fixed5. Recommend some future directions: a comparison of indoor crops and outdoor crops (outdoor crops may show a seasonal community succession), and survey the microbiome of police seizures from well-provenanced foreign sources, such as Mexico.Added to conclusion paragraph."
}
]
},
{
"id": "11800",
"date": "04 Feb 2016",
"name": "Donald Cooper",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is well written and appropriate as an article in F1000 Research. The abstract states that their findings, \"demonstrate the limitations in the culture-based regulations\", but this conclusion does not follow from their data. In fact, their results show that their DNA based method is overly sensitive at detecting potential pathogens. Whether culture-based regulations are appropriate or not would have to be validated and tested directly, not simply inferred from the presence of microbial DNA. The authors should remove this cautionary sentence in the abstract and throughout the manuscript until it has been validated. This would make the manuscript more balanced and justified.A better understanding of the microbiome and mycobiome in cannabis is an important endeavor in part, because recent work on the human microbiome has revealed that microbial constituents of the microbiome and fungi interact cooperatively and non cooperatively to influence human health. Recently studies focused on the human gut mycobiome have been performed using deep sequencing of the ITS1 region for identification of fungi in fecal samples from healthy individuals and the researchers identified 184 fungal species in total. Human oral and lung tissues testing indicate the presence of Aspergillus, Cryptococcus, Fusarium, and Alternaria in healthy individuals. An emerging theme from this new field of study indicates complex microbial communities distributed across the body that fundamentally contribute to the development, physiology and metabolic homeostasis of the macro-organism. The same is likely true in plants, like cannabis. Because of this beneficial dynamic interplay between microbes and hosts a complete absence of amplified DNA or RNA microbial markers would be unexpected. The question is, “What levels are safe?” and the answer to this question has not been established.From a consumer safety perspective the rationale for microbial testing in food and pharmaceuticals is to prevent infection of highly toxigenic microbes that grow readily and are consumed in rather large quantities. For example, Aspergillus flavus infects grain in as much as 30% of Sub-Saharan African maize and the Aflatoxin regulatory limit for maize is 10 ppb because in this region maize is consumed in kilogram quantities on a daily basis. The risk of health hazard to individuals arising from microbial toxins or mycotoxins is largely proportional to the consumption amounts and frequency. Compared to other regulated foods or tobacco it is expected that the level of cannabis consumption would be minimal and would therefore present minimal risk, nevertheless, there is burgeoning interest in putting in place regulatory requirements for medical and recreational Cannabis in some markets. In this present study the authors use state-of-the art technology to identify DNA-based markers associated with a variety of microbes and, as expected, some are pathogenic. The authors state that “Mycotoxin monitoring in Cannabis preparations is important since aflatoxin produced by Aspergillus species is a carcinogen.”, but there is no actual data showing that the strain of Aspergillus they identify is toxigenic. Aspergillus is a common fungus that is found in the human mycobiome in oral, lung, gastrointestinal tract. Detection of Aspergillus markers in minute quantities alone is not necessarily a health concern unless it is also coincident with live cells that can grow readily and secrete toxin that are above some threshold or there is an imbalance in homeostatic growth limiting factors. The authors call into question culture-based testing, which is the standard in Food safety and USP regulatory guidelines based on comparison to their highly sensitive DNA based detection. Their results using standard regulatory methods, for the most part, would not indicate a food safety problem. The authors should add this emphasis. The authors correctly identify a limitation of their study in that the qPCR based testing has an “indifference to living or non-living DNA” and because of this their PCR based approach may be unnecessarily sensitive. To date there are little to no guidelines for thresholds on many mycotoxins or bacterial toxins that have been established in the cannabis industry, so their findings help inform regulators as to which type of toxins might be relevant for further analysis. The authors state, ”Health compromised patients exposed to aflatoxin and clearance-inhibiting cannabinoids raise new questions in regards to the current safety tolerances to aflatoxin.” but the authors present no data showing the presence of aflatoxin in any of their samples. So their caution is highly speculative and they should indicate that unless they provide data supporting their caution. As mentioned above, dynamic homeostatic processes limit the growth of microbes and fungi in living organisms and the authors point out that “several studies have demonstrated plant phytochemicals and terpenoids like eugenol can inhibit the growth of fungi. It is possible the different water activity of the culture assay compared to the natural terpene rich flower environment is contributing to the false negative test results.” but these phytochemicals may also prevent the growth of fungi and bacteria in the plant despite the presence of microbial DNA. Furthermore they state that “While techniques exist to perform live-dead qPCR, the live status of the microbes is unrelated to toxin potentially produced while the microbes were alive. ELISA assays exist to screen for some toxins. Current state-recommended ELISA’s do not detect citrinin or paxilline, the toxins produced by P. citrinum and P. paxilli, respectively. The predominance of these Penicillium species in a majority of the samples tested is interesting.” Ideally the authors would test for these toxins in their most positive samples.The authors state that, “Cannabidiol is often used at micromolar concentrations for seizure reduction implying sub-percentage contamination of paxilline could still be a concern” but this is highly speculative and the authors should de-emphasize the “concern” and state instead that if their results were verified by tests indicating high levels of paxilline then it may be cause for concern. The same is true for their concern about Citrinin and aflatoxin and the authors should state this. The authors state, “While ELISA assays are easy point of use tests that can be used to detect fungal toxins, they can suffer from lack of sensitivity and cross reactivity. ITS amplification and sequencing offers hypothesis-free testing that can complement the lack of specificity in ELISA assays.” ELISA and rapid diagnostic lateral flow tests are standard in the food safety industry for measuring toxins. I see no need to call into question protein based ELISA methods without even testing them in the first place. All diagnostic tests have sensitivity and selectivity limitations which is why they need to be tested and verified using other analytical methods. The authors state that “Appropriate primer design can survey a broad spectrum of microbial genomes while affording rapid iteration of design. Quantitative PCR has also demonstrated single molecule sensitivity and linear dynamic range over 5 orders of magnitude offering a very robust approach for detection of microbial risks. This may be important for the detection of nanomolar potency mycotoxins”. The ability to detect single copies of genes makes their system highly sensitive, but does not indicate level of mycotoxin. The authors should point out the limitations of their approach and discuss the possibility that it would likely generate a high degree of false positive results compared to culture-based standard methods. The authors state, “These results demonstrate that culture based techniques superimposed from the food industry should be re-evaluated based on the known microbiome of actual Cannabis flowers in circulation at dispensaries.” This statement appears to be too strong in light of their data. Without validation for the presence of toxins above a safety threshold there is no need to re-evaluate the standard methods in the food industry.",
"responses": [
{
"c_id": "1872",
"date": "10 May 2016",
"name": "Kevin McKernan",
"role": "Author Response",
"response": "The manuscript is well written and appropriate as an article in F1000 Research. The abstract states that their findings, \"demonstrate the limitations in the culture-based regulations\", but this conclusion does not follow from their data. In fact, their results show that their DNA based method is overly sensitive at detecting potential pathogens. Whether culture-based regulations are appropriate or not would have to be validated and tested directly, not simply inferred from the presence of microbial DNA. The authors should remove this cautionary sentence in the abstract and throughout the manuscript until it has been validated. This would make the manuscript more balanced and justified. Author response: The last sentence of the abstract was revised to read: Here we describe the first next generation sequencing survey of the fungal communities found in dispensary based Cannabis flowers by ITS2 sequencing, and demonstrate the sensitive detection of several toxigenic Penicillium and Aspergillus species, including P. citrinum and P. paxilli, that were not detected by one or more culture-based methods currently in use for safety testing.A better understanding of the microbiome and mycobiome in cannabis is an important endeavor in part, because recent work on the human microbiome has revealed that microbial constituents of the microbiome and fungi interact cooperatively and non cooperatively to influence human health. Recently studies focused on the human gut mycobiome have been performed using deep sequencing of the ITS1 region for identification of fungi in fecal samples from healthy individuals and the researchers identified 184 fungal species in total. Human oral and lung tissues testing indicate the presence of Aspergillus, Cryptococcus, Fusarium, and Alternaria in healthy individuals. An emerging theme from this new field of study indicates complex microbial communities distributed across the body that fundamentally contribute to the development, physiology and metabolic homeostasis of the macro-organism. The same is likely true in plants, like cannabis. Because of this beneficial dynamic interplay between microbes and hosts a complete absence of amplified DNA or RNA microbial markers would be unexpected. The question is, “What levels are safe?” and the answer to this question has not been established.From a consumer safety perspective the rationale for microbial testing in food and pharmaceuticals is to prevent infection of highly toxigenic microbes that grow readily and are consumed in rather large quantities. For example, Aspergillus flavusinfects grain in as much as 30% of Sub-Saharan African maize and the Aflatoxin regulatory limit for maize is 10 ppb because in this region maize is consumed in kilogram quantities on a daily basis. The risk of health hazard to individuals arising from microbial toxins or mycotoxins is largely proportional to the consumption amounts and frequency. Compared to other regulated foods or tobacco it is expected that the level of cannabis consumption would be minimal and would therefore present minimal risk, nevertheless, there is burgeoning interest in putting in place regulatory requirements for medical and recreational Cannabis in some markets. In this present study the authors use state-of-the art technology to identify DNA-based markers associated with a variety of microbes and, as expected, some are pathogenic. The authors state that “Mycotoxin monitoring in Cannabis preparations is important since aflatoxin produced by Aspergillus species is a carcinogen.”, but there is no actual data showing that the strain of Aspergillus they identify is toxigenic. Aspergillus is a common fungus that is found in the human mycobiome in oral, lung, gastrointestinal tract. Detection of Aspergillus markers in minute quantities alone is not necessarily a health concern unless it is also coincident with live cells that can grow readily and secrete toxin that are above some threshold or there is an imbalance in homeostatic growth limiting factors. The authors call into question culture-based testing, which is the standard in Food safety and USP regulatory guidelines based on comparison to their highly sensitive DNA based detection. Their results using standard regulatory methods, for the most part, would not indicate a food safety problem. The authors should add this emphasis. The authors correctly identify a limitation of their study in that the qPCR based testing has an “indifference to living or non-living DNA” and because of this their PCR based approach may be unnecessarily sensitive. To date there are little to no guidelines for thresholds on many mycotoxins or bacterial toxins that have been established in the cannabis industry, so their findings help inform regulators as to which type of toxins might be relevant for further analysis. The authors state, ”Health compromised patients exposed to aflatoxin and clearance-inhibiting cannabinoids raise new questions in regards to the current safety tolerances to aflatoxin.” but the authors present no data showing the presence of aflatoxin in any of their samples. So their caution is highly speculative and they should indicate that unless they provide data supporting their caution. Author response: We thank the reviewer for the background and context provided to his concern. The questions about what levels of toxins are safe or acceptable fall well beyond the scope of the present study. We concede, however, that some statements made in the introduction may have been too speculative. We have revised paragraph 2 of the introduction to focus on the numerous literature reports of pulmonary aspergillosis associated with cannabis use instead of potential mycotoxin toxicity. We have also provided more background on the overlap of cannabinoid and mycotoxin metabolism via cytochrome P450 system as follows. Monitoring for mycotoxic fungi in cannabis preparations has been recommended as part of routine safety testing by the Cannabis Safety Institute. A major driver for this recommendation has been numerous reported cases of serious or fatal pulmonary Aspergillosis associated with marijuana smoking in immunocompromised patients4-6. The major cannabinoids have been shown to be potent inhibitors of several cytochrome P450 enzymes at therapeutic concentrations, including 1A1, 1A2, 1B1 2B6, 2C19, 2D6, 3A4 and 3A57. Some of these enzymes have been implicated in the metabolism of the fungal toxins aflatoxin and ochratoxin8-10. This raises questions about potential interactions and appropriate safety tolerances for mycotoxins in patients being treated with cannabinoid therapeutics. In addition, some Fusarium species that produce toxins have proven to be difficult to selectively culture with tailored media 6– 8 . This is a common problem associated with culture-based systems as carbon sources are not exclusive to certain microbes and only 1% of microbial species are believed to be culturable 9 . The first sentence of the next paragraph was edited slightly to follow more logically: While the risks of mycotoxic fungal contamination have been well studied in the food markets, the presence of the fungal populations present on Cannabis flowers has never been surveyed with next generation sequencing techniques 10– 15 .As mentioned above, dynamic homeostatic processes limit the growth of microbes and fungi in living organisms and the authors point out that “several studies have demonstrated plant phytochemicals and terpenoids like eugenol can inhibit the growth of fungi. It is possible the different water activity of the culture assay compared to the natural terpene rich flower environment is contributing to the false negative test results.” but these phytochemicals may also prevent the growth of fungi and bacteria in the plant despite the presence of microbial DNA. Author response: Upon reconsideration, we concluded that there is no way to predict how growth of the specific fungi detected might be affected by the terpenoids in Cannabis flowers. Those compounds may inhibit the growth of some fungi, while permitting the growth of others. In any case, the effect of terpenes would not relevant to the ability of the fungi to grow on culture media lacking those compounds. That sentence was deleted and the last sentence of paragraph 1 in the discussion was edited as follows: It is possible the different water activity of the culture assay compared to the natural flower environment is contributing to the false negative test results.Furthermore they state that “While techniques exist to perform live-dead qPCR, the live status of the microbes is unrelated to toxin potentially produced while the microbes were alive. ELISA assays exist to screen for some toxins. Current state-recommended ELISA’s do not detect citrinin or paxilline, the toxins produced by P. citrinum and P. paxilli, respectively. The predominance of these Penicillium species in a majority of the samples tested is interesting.” Ideally the authors would test for these toxins in their most positive samples. Author response: We stated transparently that this needed to be done as a future direction. We don’t see it as an essential part of the current publication, which had the goal of simply cataloging the fungal content of dispensary-derived samples.The authors state that, “Cannabidiol is often used at micromolar concentrations for seizure reduction implying sub-percentage contamination of paxilline could still be a concern” but this is highly speculative and the authors should de-emphasize the “concern” and state instead that if their results were verified by tests indicating high levels of paxilline then it may be cause for concern. The same is true for their concern about Citrinin and aflatoxin and the authors should state this. We agree. Paragraph 3 of the discussion was edited to address this criticism as follows: Paxilline is a tremorgenic and ataxic potassium channel blocker and has been shown to attenuate the anti-seizure properties of cannabidiol in certain mouse models 44– 46 . Paxilline is reported to have tremorgenic effects at nanomolar concentrations and is responsible for Ryegrass-staggers disease 47 . Cannabidiol is often used at micromolar concentrations for seizure reduction and contamination with paxilline, if confirmed, would be a cause for concern. Citrinin is a mycotoxin that disrupts Ca2+ efflux in the mitochondrial permeability transition pore (mPTP) 48– 55 . Ryan et al. demonstrated that cannabidiol affects this pathway suggesting a similar potential cause for concern regarding CBD-citrinin interaction 56 . Considering the hydrophobicity of these mycotoxins and the growing interest in the use of extracted oils from CBD-rich Cannabis strains for treatment of drug resistant epilepsy57– 62, more precise molecular screening of fungal toxins in these products might be warranted.The authors state, “While ELISA assays are easy point of use tests that can be used to detect fungal toxins, they can suffer from lack of sensitivity and cross reactivity. ITS amplification and sequencing offers hypothesis-free testing that can complement the lack of specificity in ELISA assays.” ELISA and rapid diagnostic lateral flow tests are standard in the food safety industry for measuring toxins. I see no need to call into question protein based ELISA methods without even testing them in the first place. All diagnostic tests have sensitivity and selectivity limitations which is why they need to be tested and verified using other analytical methods. The authors state that “Appropriate primer design can survey a broad spectrum of microbial genomes while affording rapid iteration of design. Quantitative PCR has also demonstrated single molecule sensitivity and linear dynamic range over 5 orders of magnitude offering a very robust approach for detection of microbial risks. This may be important for the detection of nanomolar potency mycotoxins”. The ability to detect single copies of genes makes their system highly sensitive, but does not indicate level of mycotoxin. The authors should point out the limitations of their approach and discuss the possibility that it would likely generate a high degree of false positive results compared to culture-based standard methods. Author response: The comments about ELISA assays was deleted and the paragraph was edited to focus on detection of fungal species, not toxins, as follows. ITS amplification and sequencing offers a hypothesis-free testing approach that can be employed to identify a broad range of fungal species present in a given sample. Appropriate primer design can survey a broad spectrum of fungal genomes while affording rapid iteration of design. Quantitative PCR has also demonstrated single molecule sensitivity and linear dynamic range over 5 orders of magnitude offering a very sensitive approach for detection of microbial risks. Our survey of Cannabis flowers in this study was limited, however. Further studies are required to survey a broader range of samples, and to determine whether paxilline, citrinin, aflatoxin or ochratoxin can be detected at concentrations that represent a clinical risk in Cannabis samples or extracts derived from plants that test positive for the fungi known to produce those toxins.The authors state, “These results demonstrate that culture based techniques superimposed from the food industry should be re-evaluated based on the known microbiome of actual Cannabis flowers in circulation at dispensaries.” This statement appears to be too strong in light of their data. Without validation for the presence of toxins above a safety threshold there is no need to re-evaluate the standard methods in the food industry.Author response: We respectfully disagree with this comment of the reviewer. The sentence was taken out of context. We were not trying to suggest that the standard methods in use in the food industry should be re-evaluated for all applications, only the use of those methods for medicinal Cannabis testing. The Conclusions paragraph was modified to clarify as follows. Several toxigenic fungi were detected in dispensary-derived Cannabis samples using molecular amplification and sequencing techniques. These microbes were not detected using traditional culture-based platforms. These results suggest that culture based techniques borrowed from the food industry should be re-evaluated for Cannabis testing to ensure that they are capable of detecting the prevalent species detected by molecular methods with adequate sensitivity. We recommend that additional sequencing studies be performed to characterize the fungal and bacterial microbiomes of a more diverse selection of Cannabis samples. Such sampling should include dispensary-derived samples from both indoor and outdoor crops, as well as samples from police seizures from well-provenanced foreign sources, such as Mexico. Finally, further studies should be performed to measure toxin levels in strains that test positive for toxigenic species."
}
]
}
] | 1
|
https://f1000research.com/articles/4-1422
|
https://f1000research.com/articles/5-862/v1
|
10 May 16
|
{
"type": "Case Report",
"title": "Case Report: Successful delivery in a 44-year-old woman using vitrified human oocytes taken from the woman at 41 years of age",
"authors": [
"Miyako Funabiki",
"Yoshitaka Nakamura",
"Sagiri Taguchi",
"Miyako Funabiki",
"Yoshitaka Nakamura"
],
"abstract": "Successful pregnancies and deliveries are rare in women over 40 years of age. To date, no case report has been published about a successful delivery in a woman over 40 years of age using vitrified oocytes obtained through social egg freezing from a woman over 40 years of age. Here we report a case of a successful live birth from a 44-year-old woman using vitrified oocytes taken from the woman at 41 years of age; the patient used the technique for social reasons. The 44-year-old patient delivered a 2534 g female infant by Caesarean section in the 37th week of pregnancy. The female infant’s Apgar scores were 9 at 1 min and 9 at 5 min. The female infant exhibited no anomalies and is developing normally under the close surveillance of pediatricians.However, the concept of social egg freezing has medical, ethical and social problems. Furthermore, the rate of live births differs among clinics and/or hospitals due to variations in the vitrification and warming techniques used. Therefore, information from individual clinics and/or hospitals about the risks, the rates of successful live births and the cost of social egg freezing should be provided to patients to aid in the patient’s decision-making process.",
"keywords": [
"Women over 40 years old",
"vitrified human oocytes",
"social egg freezing",
"live birth"
],
"content": "Introduction\n\nGiven the advancement in technology for human egg freezing, the use of social egg freezing is increasing in developed countries1,2. The increase in social egg freezing is associated with a number of different personal, professional, economical and psychological reasons1,2.\n\nHowever, successful pregnancies and deliveries are rare in women over 40 years of age1,2. Furthermore, no case report about a successful delivery from a woman over 40 years of age using vitrified oocytes obtained through social egg freezing from a woman over 40 years of age has been published to date (as assessed through a PubMed database search).\n\n\nCase\n\nThe patient visited our in vitro fertilization (IVF) clinic in April 2012. The patient was a 41-year-old woman who wanted to freeze her eggs for social reasons. She provided written informed consent. From April 2012 to June 2013, 8 oocytes were vitrified through ultra-rapid cooling using a high concentration of cryoprotectants (15% ethylene glycol + 15% DMSO + 0.5 mol/l sucrose) in a cryo device (Vitrification Media Kit, KITAZATO CORPORATION, Tokyo, Japan) by plunging the oocytes into liquid nitrogen (-196°C). The 8 oocytes were cryopreserved within liquid nitrogen (-196°C). The vitrified oocytes were thawed at the time of use. In June 2014, two embryos (day 2; a vitrified oocyte-derived embryo and a standard-derived embryo) were transferred to the uterus. The embryo transfers were carried under ultrasound guidance when endometrium thickness reached 7.0 mm. However, the patient’s pregnancy was not confirmed by the presence of a gestational sac (GS) at this time.\n\nIn August 2014, an embryo (day 5) from a vitrified oocyte was transferred to the uterus (the endometrium thickness: 7.5 mm), and the patient’s pregnancy was confirmed by the presence of a GS at 5 weeks (Figure 1). Repeated ultrasonography during the pregnancy revealed normal fetal growth and development. The 44-year-old patient delivered a 2534 g female infant by Caesarean section in the 37th week of pregnancy in May 2015. The female infant’s Apgar scores were 9 at 1 min and 9 at 5 min. The female infant exhibited no anomalies and is developing normally under the close surveillance of pediatricians.\n\n\nDiscussion\n\nHuman oocyte vitrification holds great promise for women who need to preserve their fertility due to cancers that require chemotherapy or radiation1,2. This technique is also referred to as “social egg freezing,” and many women who wish to delay pregnancy and/or delivery to pursue educational or professional goals want to have this option available to them1,2. However, the guidelines of the American Society for Reproductive Medicine (ASRM) caution against the use of oocyte vitrification to circumvent the effects of age on the reproductive potential of healthy women given that there are insufficient data to support the safety, efficacy, ethics, emotional risks and cost-effectiveness of oocyte cryopreservation3. In 2013, the Japan Society for Reproductive Medicine (JSRM) issued a guideline allowing egg freezing for women under 404. In addition, in 2015, an expert panel from the Japan Society of Obstetrics and Gynecology (JSOG) said it is not recommended that young and healthy women preserve their frozen eggs for future pregnancies and deliveries4,5.\n\nFurthermore, after performing a PubMed database search, no case reports have been published demonstrating the safety and efficacy of oocyte cryopreservation in women over 40 years of age who used the technique for social reasons. Therefore, we report a case of a successful live birth from a 44-year-old woman using vitrified oocytes obtained through social egg freezing from the woman at 41 years of age. Although the oocyte-to-baby rate was 1.8% and the number of oocytes typically needed to achieve a baby in women over 40 years of age is 55.56, we were fortunately able to obtain a successful example. However, the concept of social egg freezing has medical, ethical and social problems. In general, a woman’s ovarian reserve declines as she ages, resulting in fewer oocytes and an increased risk of embryonic aneuploidy1. Although we do not necessarily recommend having a child in the later stages of life, many patients who visit our IVF clinic are older than 40 years of age. Therefore, careful counseling is required because the patients must understand the uncertainties regarding the safety and efficacy of social egg freezing.\n\nIn addition, the rates of live births differ among clinics and/or hospitals due to variations in the vitrification and warming techniques used1. Therefore, information from individual clinics and/or hospitals about the risks, the rates of successful live births and the cost of social egg freezing should be provided to patients to aid in the patient’s decision-making process.\n\n\nConclusion\n\nWe report a successful live birth from a 44-year-old woman using vitrified oocytes obtained through social egg freezing from the woman when she was 41 years old. However, the concept of social egg freezing is controversial. Therefore, information concerning this process should be disclosed to aid in the patient’s decision-making process.\n\n\nConsent\n\nWritten informed consent for publication of the clinical details and clinical images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nAll authors equally contributed to the writing of this 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\nSchattman GL: CLINICAL PRACTICE. Cryopreservation of Oocytes. N Engl J Med. 2015; 373(18): 1755–1760. PubMed Abstract | Publisher Full Text\n\nPetropanagos A, Cattapan A, Baylis F, et al.: Social egg freezing: risk, benefits and other considerations. CMAJ. 2015; 187(9): 666–669. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPractice Committees of American Society for Reproductive Medicine; Society for Assisted Reproductive Technology: Mature oocyte cryopreservation: a guideline. Fertil Steril. 2013; 99(1): 37–43. PubMed Abstract | Publisher Full Text\n\nEditorials.: Egg-freezing: choice and risk. The Japan Times. 2016. Reference Source\n\nKyodo.: Japanese woman has baby through egg freezing, defying expert guidance. The Japan Times. 2016. Reference Source\n\nCobo A, Garrido N, Pellicer A, et al.: Six years' experience in ovum donation using vitrified oocytes: report of cumulative outcomes, impact of storage time, and development of a predictive model for oocyte survival rate. Fertil Steril. 2015; 104(6): 1426–34.e1–8. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "14280",
"date": "10 Jun 2016",
"name": "Charles Coddington",
"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\nTitle and Abstract: The title is reflective of the content of the article. The abstract adequately summarizes the article.\n\nArticle content: The topic of the case report is very interesting. The authors can expand the current body of knowledge on elective fertility preservation in this age group by providing more details about the patient. For instance demographic information such as the patient’s gravity, parity, medical comorbidities, BMI, ovarian reserve (Day 3 FSH, E2, AMH, antral follicle count) are missing. It can be inferred that the patient underwent more than one stimulation cycle. The number of cycles she underwent to reach 8 oocytes and the protocol is also omitted. The grade of the embryos transferred was also not shared. Most importantly the indication for the early term c-section at 37 weeks and any maternal medical conditions that developed antenatal, intra-partum or post-partum are extremely relevant and should be reported. Providing more information would allow the information to be applied in similar scenarios.\n\nConclusions: The conclusion was well thought out. However, given very little information was given about the patient is difficult to assess if alternative conclusions would be more plausible.",
"responses": []
},
{
"id": "14253",
"date": "20 Jun 2016",
"name": "Robert Casper",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript the authors present a case report of social oocyte vitrification in a woman at age 41 that resulted in a live birth when the oocytes were warmed and fertilized 3 years later. This may be the first reported case of successful pregnancy from vitrified oocytes over age 40.\n\nIn my opinion, this case adds nothing new to the literature. There is no new technology nor surprising new findings since standard vitrification techniques and routine in vitro fertilization were used in this patient. The unusual finding is that she achieved a successful pregnancy.\n\nIn contrast, this case report may raise false hope for other women over age 40 who have delayed pregnancy and think that social egg freezing may be an option to preserve their fertility. As the authors themselves point out, the clinic that perfected vitrification of oocytes and that has the most experience with the technique, estimated that 55 oocytes would have to be vitrified in a women over age 40 to achieve a pregnancy and live birth (Cobo et al, 2015). I believe that this number should be enough to discourage any woman from considering social or other oocyte freezing over age 40, or any clinic from offering this procedure to women over 40. In our clinic, we have an upper age limit for social age freezing of 39 years, although I am aware of other clinics in which this is not the case.\n\nThe patient in the present case report was lucky to have a euploid oocyte that survived, was fertilized and implanted allowing her to conceive. In the course of a year, from April 2012 to June 2013 she had 8 oocytes vitrified. Presumably it required several cycles to obtain the 8 oocytes and the authors should provide this information. The first embryo transfer in which the patient did not conceive included an embryo derived from one of the vitrified oocytes and one from a fresh IVF cycle, suggesting that the woman was still ovulating. The authors need to describe the endometrial preparation technique used for the successful embryo transfer. Was it an exogenous estrogen and progesterone hormonal preparation of the endometrium or a natural cycle? If the latter, how did the authors ensure that the pregnancy was not achieved as a result of natural conception of an ovulated oocyte?",
"responses": []
},
{
"id": "15656",
"date": "12 Aug 2016",
"name": "Laxmi Baxi",
"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\nTitle is appropriate and indicates what the substance of the case report is.\nUnfortunately, there are certain issues need to be addressed.\nAs expected, the likelihood of a successful pregnancy in a woman the age of 40 years and above would be low. Authors need to address the issue as to which individuals are likely to have a successful pregnancy. Genetic, environmental and hormonal milieu and family history, age at menopause in other females in the family need to be addressed.\nThere is no case report in the literature - PubMed - as it is not impossible for women to have a successful pregnancy after the age of 40 yrs of age and hence not a rare event.\nIt would be worthwhile if authors were to analyse all these factors and genetic studies if possible to describe factors contributing to successful egg donation and pregnancy thereafter.\nLastly, why did this patient undergo caesarean delivery to deliver her baby? This is an unanswered question.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-862
|
https://f1000research.com/articles/5-381/v2
|
10 May 16
|
{
"type": "Software Tool Article",
"title": "pyGeno: A Python package for precision medicine and proteogenomics",
"authors": [
"Tariq Daouda",
"Claude Perreault",
"Sébastien Lemieux",
"Claude Perreault"
],
"abstract": "pyGeno is a Python package mainly intended for precision medicine applications that revolve around genomics and proteomics. It integrates reference sequences and annotations from Ensembl, genomic polymorphisms from the dbSNP database and data from next-gen sequencing into an easy to use, memory-efficient and fast framework, therefore allowing the user to easily explore subject-specific genomes and proteomes. Compared to a standaloneprogram, pyGeno gives the user access to the complete expressivity of Python, a general programming language. Its range of application therefore encompasses both short scripts and large scale genome-wide studies.",
"keywords": [
"Bioinformatics",
"python",
"database",
"precision medicine",
"genomics",
"proteomics",
"proteogenomics",
"genetic polymorphisms"
],
"content": "Introduction\n\nHigh-throughput systems biology and precision medicine applications require the integration of data from many different sources. For instance, a significant part of precision medicine research revolves around the identification of relevant single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELS) and the study of their context1. Furthermore recent studies in proteogenomics show that replacing traditional reference databases such as Uniprot2 by customized databases that integrate the subject’s genomic polymorphisms, can significantly improve the identification of peptides or proteins using mass spectrometry3–6. These applications usually require the integration of reference sequences, reference genome annotations, specific SNPs and INDELs along with an external SNP database such as dbSNP7 for validation. The sheer amount of data generated by theses studies rules out most spreadsheet analyses and requires tools that are both fast and memory efficient. Furthermore, these studies often require the collaboration of people with different sets of skills. Thus, it was important to us to develop a tool that is powerful enough to be integrated in complex high-throughput pipelines, while still being understandable by users with limited technical abilities. In contrast to other projects such as BioPython8 and PyCogent9 whose objective is to provide a general set of tools for bioinformatics, the primarily ambition behind pyGeno is to provide the community with a powerful genome and proteome exploration tool that can be easily integrated into scripts. The current version integrates gene set annotations and reference sequences from Ensembl10 along with polymorphisms (both SNPs and INDELs) derived from dbSNP7, and experimentally detected patient-specific polymorphisms.\n\nTo our knowledge pyGeno is the only available tool that provides this kind of integration in an easy-to-use and programming-friendly environment. Furthermore, more advanced users can rely on object-oriented inheritance to extend the functionalities of pyGeno to implement support for polymorphisms from other sources. pyGeno has been used with human and mouse genomes and should readily work with any diploid organism whose annotations are made available by Ensembl.\n\n\nMethods\n\npyGeno is written in Python, a language that enjoys a large set of well established and mature scientific libraries that are used in research fields such as physics, mathematics and bioinformatics8,11–13. pyGeno gives users access to the full expressivity of Python to explore reference and patient-specific genomes and proteomes, by manipulating familiar objects such as genomes, chromosomes, genes, transcripts, proteins and exons. In order to make pyGeno as easy to use and learn as possible, we have created an interface where only one function, get(), can be used for almost any query. An example of usage can be seen in Figure 1. An integrated documentation is also available through the help() function.\n\nThe current version of pyGeno does not require any access to remote REST APIs. This results in more robust and faster processing since the application is not affected by connection speed or sudden changes to the server API. On the other hand it also implies that extra care must be taken regarding the optimization of the application.\n\n(A) Here we instantiate a personalized genome G1 by providing the Genome constructor with the name of a reference genome, a set of polymorphisms and a user defined SNP filter (for example a quality filter). (B) We then ask the get function of G1 to return a protein by id. The result is an object where only the fields in bold are fully loaded, other fields will be automatically loaded when and if accessed. (C) Asking for the currently unloaded sequence of the protein triggers the following sequence of events. The transcript, as well as the exons that encode for it, and any polymorphisms in their regions are loaded. The polymorphisms are filtered according to the filter provided to the genome constructor (for example, according to sequencing quality) and inserted at their corresponding locations. The exons are then assembled into the transcript sequence and the sequence is translated. (D) The sequence as well as the transcript are now fully loaded and the sequence of the precision protein is printed.\n\nMemory efficiency and speed are mainly achieved through the use of a custom lazy object-oriented database system that we have specifically written for pyGeno (https://github.com/tariqdaouda/rabaDB). When an object is loaded through the get() function, only a minimal version of it is served. The object fully develops only once the user accesses a field that is not present in the minimal version (Figure 1). The transformation is entirely transparent and does not require more memory than necessary to store the fully developed object. This is especially important, since most of the time users are only interested in specific regions of the genome, and do not require that the full genome be loaded into memory. Every loaded object is also a singleton, if the user asks for a previously loaded object, pyGeno will serve the object in memory.\n\nFurthermore, this database system is built on top of SQLite version 3 (http://www.sqlite.org/), a serverless relational database. Because SQLite3 uses single files to store data, pyGeno’s database can be easily backed up and shared by a simple copy/paste. Moreover, the files can be directly read, modified and analyzed through any SQLite3 client.\n\nAs with any other database system, indexes play a crucial role in determining the general performance. Within pyGeno’s database, several reference genomes along with patient-specific data and versions of dbSNP can coexist. Therefore building indexes for all the stored information would result in unnecessarily large databases. We therefore have taken the approach of giving the end user full control over indexation through the ensureGlobalIndex() and dropGlobalIndex() functions. Users can, for example, decide to index the field ’id’ of transcripts by using Transcript.ensureGlobalIndex(’id’) and dramatically improve queries based on transcript ids.\n\npyGeno’s database is populated through imports of datawraps using importSNPs and importGenome functions. Datawraps are compressed archives that can be shared among co-workers, and are designed to solve the version and update problems. A datawrap contains at least one file named manifest.ini that contains basic information about the package such as a description, a version and a maintainer, as well a list of files from which data must be extracted. It is possible to either compress these files within the archive, or to specify URLs from which the files can be downloaded.\n\nIn an effort to make pyGeno as easy to install as possible we have made it as dependency-free as possible. This approach has motivated our choice for SQLite3, since it is natively supported by Python 2.5 and above, and it also lead us to develop many tools that were subsequently integrated into pyGeno. Among theses tools are various functions for translating sequences, parsers for GTF/GFF, VCF, FASTA, FASTQ and CSV files, a progress bar, and an efficient way of annotating the genome called segment trees.\n\n\nPersonalized genomes\n\nOne of the biggest strengths of pyGeno is to allow the user to define personalized genomes. These genomes are built by combining a reference genome with sets of polymorphisms and a filtering function that returns the alleles to be inserted at the appropriate locus (Figure 1). Personalized genomes are a powerful tool that can go beyond the definition of patient-specific genomes. For instance, we recently used this tool to combine the results of both RNA- and DNA-seq data and create more robust personalized genomes that were used to identify protein-derived peptides by mass spectrometry3. Furthermore because pyGeno loads the necessary parts of a given reference genome only once, a pyGeno application can handle several personalized genomes without significantly increasing its memory consumption.\n\npyGeno’s only requirement is Python2 and we highly recommend version 2.7.6 or later. pyGeno can be easily installed using the pip package manager (https://pip.pypa.io/) by typing pip install pyGeno into command line interface. Alternatively the latest developments can be obtained from the github repository. Once pyGeno’s installation has been completed, the first action that users must perform is the importation of a reference genome datawrap. In order to simplify the process pyGeno comes with several datawraps that can be directly listed and installed using its bootstrap module. If the desired reference genome is not among the ones provided, users also have the possibility to create their own from scratch by following the steps described in the documentation. After the first reference genome importation, pyGeno is fully functional and users can further expand its database by importing other reference genomes or SNP sets.\n\n\nSummary\n\nWe have developed pyGeno because, in an age where both precision medicine and DNA/RNA sequencing are becoming more and more important, we needed a tool that would allow us to easily work on personalized genomes that include subject-specific genomic features. Nowadays research teams are increasingly multidisciplinary and are composed of people with very different backgrounds. Since we wanted pyGeno to serve as a common language between users, we therefore took great care in making pyGeno easy to install, easy to use and optimized it so it can run on computers with limited resources (eg. laptops). The fact that pyGeno has been downloaded more than 12,000 times over its first year of existence suggests that there is indeed a need for powerful user-friendly precision medicine tools. With pyGeno we have taken a rather unusual approach to user-friendliness. Instead of writing a program with a graphical user interface (GUI), we have decided to create a Python module that fully integrates within the Python environment. This ensures that users can leverage the full expressiveness of Python as well as the functionalities of other python modules such as SciPy and numpy11, pandas (http://pandas.pydata.org/) and matplotlib13, to meet their specific needs. Furthermore, it led us to think of the functions and objects the user manipulates as pyGeno’s interface and we strived to make it as simple and easy to learn as possible.\n\nIn the past few years great technologies have been developed. Scripting languages such as Python and JavaScript have taken programming to a whole new level of simplicity, and are now fast enough to serve as foundations to large-scale projects. Freely available libraries such as D3.js (http://d3js.org/) allow for the creation of stunning data representations, that once coupled with tools such as pyGeno, could be used to create powerful interactive representations of biological data. The NoSQL movement has produced several new database systems from which developers can choose, offering them the opportunity to store sheer amounts of data with a flexibility that was not present only a few years ago. These technologies and many others are only waiting to be put together into ground breaking tools for the treatment of biological data. In life saving research areas, we believe that great tools that dramatically improve workflow efficiency are not a luxury but a necessity.\n\n\nSoftware availability\n\n\n\n1. pyGeno is available from the Python Package Index (PyPI; https://pypi.python.org) via: pip install pyGeno.\n\n2. Latest source code: https://github.com/tariqdaouda/pyGeno.\n\n3. Documentation: http://pyGeno.iric.ca\n\n4. Link to archived source code as at time of publication: https://zenodo.org/record/50587#.VyIP0UErJB0 (doi:10.5281/zenodo.50587)\n\n5. License: Apache License Version 2.0",
"appendix": "Author contributions\n\n\n\nTD designed and developed pyGeno. SL, CP, and TD contributed to the preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Canadian Cancer Society (Grant number 701564), assigned to Claude Perreault.\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 Jean-Philippe Laverdure, Céline Laumont and Hillary Pearson for being the first users (outside of the developer) and the first testers of pyGeno.\n\n\nReferences\n\nCollins FS, Varmus H: A new initiative on precision medicine. N Engl J Med. 2015; 372(9): 793–795. PubMed Abstract | Publisher Full Text\n\nUniprot Consortium: Update on activities at the Universal Protein Resource (UniProt) in 2013. Nucleic Acids Res. 2013; 41(Database issue): D43–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGranados DP, Sriranganadane D, Daouda T, et al.: Impact of genomic polymorphisms on the repertoire of human MHC class I-associated peptides. Nat Commun. 2014; 5: 3600. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim MS, Pinto SM, Getnet D, et al.: A draft map of the human proteome. Nature. 2014; 509(7502): 575–581. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilhelm M, Schlegl J, Hahne H, et al.: Mass-spectrometry-based draft of the human proteome. Nature. 2014; 509(7502): 582–587. PubMed Abstract | Publisher Full Text\n\nLaumont CM, Daouda T, Laverdure JP, et al.: Global proteogenomic analysis of human MHC class I-associated peptides derived from non-canonical reading frames. Nat Commun. 2016; 7: 10238. PubMed Abstract | Publisher Full Text\n\nSherry ST, Ward MH, Kholodov M, et al.: dbSNP: the NCBI database of genetic variation. Nucleic Acids Res. 2001; 29(1): 308–311. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCock PJ, Antao T, Chang JT, et al.: Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics. 2009; 25(11): 1422–1423. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnight R, Maxwell P, Birmingham A, et al.: PyCogent: a toolkit for making sense from sequence. Genome Biol. 2007; 8(8): R171. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlicek P, Amode MR, Barrell D, et al.: Ensembl 2014. Nucleic Acids Res. 2014; 42(Database issue): D749–D755. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones E, Oliphant T, Peterson P, et al.: SciPy: Open source scientific tools for Python. 2001; [Online; accessed 2016-02-22]. Reference Source\n\nSymPy Development Team: SymPy: Python library for symbolic mathematics. 2014. Reference Source\n\nHunter JD: Matplotlib: A 2d graphics environment. Comput Sci Eng. 2007; 9(3): 90–95. Publisher Full Text"
}
|
[
{
"id": "13874",
"date": "31 May 2016",
"name": "Lynn Fink",
"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\npyGeno is a Python package that allows a user to simply query either a standard reference genome or a custom genome for information such as sequences, SNPs, and related RNA and protein data. I agree that this package fills a need for genome research, appears to be very straightforward to use, and I would like to use it myself.\nHowever, when I tested it I was unable to get it to run; contrary to the authors claims that it is easy to install with minimal dependencies I couldn't make it work. (I am not a Python expert, but I have installed much more complicated packages successfully.) With Python virtual environments, I wonder if there is a need to be quite so minimal. Would it be easier to rely on standard packages to ensure a universal, smooth experience?",
"responses": [
{
"c_id": "2010",
"date": "06 Jun 2016",
"name": "Tariq Daouda",
"role": "Author Response",
"response": "Thank you for taking the time to review our work. In light of your review we have retested the installation, genome importation and polymorphism insertion on Linux, MacOS and Windows and it seems to work on every platform. We are fully committed to make the experience for the end user as smooth and easy as possible, if you could give us more details about the problems you encountered by filling a GitHub issue, we would be very happy to address them."
}
]
},
{
"id": "14063",
"date": "31 May 2016",
"name": "Hilary Ann Coller",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 manuscript describes the development of a valuable new Python package that provides the user with an environment in which they can explore multiple different large datasets related to a single gene. The software maps genes back to Ensemble, provides polymorphisms from dbSN, and provides information on experimentally detected patient-specific polymorphisms. The software can also handle DNA Sequencing data, RNA-Seq data and proteomics data for the same individual. The authors have designed the software so that it manages these datasets efficiently, thus providing the user with seamless and rapid access to the information desired. The use of “datawraps” so that co-authors can share information will likely also be valuable for users. The software has been downloaded over 12,000 times in the first year, demonstrating its utility. There is a very helpful figure, Figure 1, that gives an overview of the process. The authors might consider adding a section of the manuscript in which they walk the reader through the analysis of an actual gene in an actual genome to give the reader a sense of the findings.",
"responses": []
},
{
"id": "15427",
"date": "13 Sep 2016",
"name": "Christian Cole",
"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\nDaouda et al. propose a new tool, pyGeno, for the interrogation of proteomics data in the context of genomic sequence variants.\n\nGeneral Comment:\n\nSeveral times the authors make reference to ‘precision medicine’ without clarifying what is meant by the term. If the authors have a specific workflow or use case which befits ‘precision medicine’ they need to make it clearer. I note that the authors do not include a ‘Use Cases’ section as suggested by in the ‘Instructions to Authors’. Their previous paper (Granados et al, 2014) would be a great example of how to use pyGeno on real data.The examples provided in the paper are either too vague or too simplistic.\n\nToo often new tools come out which try to reinvent the wheel for a small incremental improvement. Here, the authors need to be acknowledged for using well-established systems like Ensembl, SQLite and reading in existing data types (e.g. GFF, VCF, fasta).\n\nThe paper is too short on specifics and somewhat unstructured. The Methods section is fine although would benefit from an overview- with a Figure and/ or text- as the overall structure of pyGeno is unclear. The Personalized genomes section should be expanded as a ‘Use Cases’ section. The ‘Operation’ section should be part of the Methods.\n\nThe online instructions are quite clear for installation, however I gave up due to the incredibly slow progress: >20 hours remaining of a full genome ‘datawrap’. Thus, unfortunately, I was not able to test the software myself. Given that pyGeno has been downloaded many times, this might an issue local to me.\n\nOverall I do feel pyGeno is a valuable contribution to the community, however the paper needs some improvement to highlight the tool’s usefulness better.\n\nSpecific Comments:\n\nIn the third paragraph of the Methods, the authors state that pyGeno is not dependent on any ‘remote REST APIs’. If this is the case how does the pyGeno interact with Ensembl and keep in sync with the regular updates (every 6 months)? The focus on ‘robust and faster processing’ is understandable, but version drift from official sources can be a serious problem. Is version maintenance something end users can do or are they dependent on the authors keeping the versions up-to-date?\n\nIn Figure 1, a simple example is provided showing a protein sequence with what appears to be two non-synonymous variants highlighted in the protein sequence. How does pyGeno cope with summarising/ visualising; 1) mutually exclusive variants at a single amino acid (e.g. two non-synonymous variants at different positions of the codon ), or 2) more complex variants like splicing-affecting changes and loss/gain of STOP codons? Similarly does pyGeno accept phased haplotypes thereby allowing inspection of both protein products from each of the individual’s alleles?\n\nWhat are the hardware requirements for running pyGeno and associated analyses? Is a well-specified workstation with several GB of RAM, fast cpu and terabytes of diskspace required or can it be run on a laptop?\n\nThe final paragraph does not contribute anything to the paper, I suggest the authors remove it and end the article with something more succinct and pertinent.",
"responses": []
}
] | 2
|
https://f1000research.com/articles/5-381
|
https://f1000research.com/articles/5-859/v1
|
10 May 16
|
{
"type": "Review",
"title": "Hox genes and evolution",
"authors": [
"Steven M. Hrycaj",
"Deneen M. Wellik",
"Deneen M. Wellik"
],
"abstract": "Hox proteins are a deeply conserved group of transcription factors originally defined for their critical roles in governing segmental identity along the antero-posterior (AP) axis in Drosophila. Over the last 30 years, numerous data generated in evolutionarily diverse taxa have clearly shown that changes in the expression patterns of these genes are closely associated with the regionalization of the AP axis, suggesting that Hox genes have played a critical role in the evolution of novel body plans within Bilateria. Despite this deep functional conservation and the importance of these genes in AP patterning, key questions remain regarding many aspects of Hox biology. In this commentary, we highlight recent reports that have provided novel insight into the origins of the mammalian Hox cluster, the role of Hox genes in the generation of a limbless body plan, and a novel putative mechanism in which Hox genes may encode specificity along the AP axis. Although the data discussed here offer a fresh perspective, it is clear that there is still much to learn about Hox biology and the roles it has played in the evolution of the Bilaterian body plan.",
"keywords": [
"homeobox",
"AP patterning",
"hox function",
"regionalization",
"deregionalized"
],
"content": "Introduction\n\nHox proteins are a group of homeodomain-containing transcription factors that are renowned for their roles in patterning animal body plans and for their remarkably deep evolutionary conservation. Homeodomain proteins are defined by the presence of a highly conserved DNA-binding region known as the homeodomain and are encoded by Homeobox genes. In general, homeobox genes are a large family of similar genes and can be divided into 11 different gene classes in animals, and the Hox genes belong to the ANTP class1,2. This class of genes also includes the closely related ParaHox genes, NK genes, and various others. It has been suggested that the evolution and expansion of Hox genes have played a key role in the rapid diversification of the body plans of all Bilaterians. Thus, this group of genes has fascinated evolutionary biologists for decades and continues to be studied by many research groups today.\n\nHox genes were originally discovered in Drosophila and functional studies in the fly showed that these genes play a critical role in establishing segmental identity along the antero-posterior (AP) axis3. Subsequent analyses have shown that the role of Hox genes in establishing AP axis identity is conserved in vertebrates4–6. These data were very exciting and confirmed that their function was conserved in evolutionarily distant taxa. Since their original discovery in the fly over 30 years ago, Hox genes have now been cloned and analyzed in a wide array of animal groups ranging from hydra to humans. Collectively, these studies have provided key insights into the evolutionary origins of Hox genes and have reinforced the important role these genes have played in the evolution of Bilaterian body plans.\n\nIn this review, we provide a commentary on the recent advances on the origin, functional conservation, and regulative properties of Hox genes. The purpose of this review is not to provide a comprehensive detailed survey of the literature to date but rather to highlight recent data that have both challenged traditional views and enhanced our understanding of Hox genes and evolution.\n\n\nEvolution of the Hox genes\n\nNone of the ANTP class of homeobox genes (including the Hox genes) is found outside of the metazoans2. During the evolution of metazoans, the sponges diverged first, followed by cnidarians (jellyfish and corals), and both of these groups are more basal to the Bilaterians. Analysis of whole genome information from the demosponge Amphimedon queenslandica revealed the first conclusive evidence that sponges have several NK homeobox genes but do not have any definitive Hox or ParaHox genes7. In contrast, definitive Hox-like genes have been identified in the Cnidarians; however, the expression patterns of these genes do not follow a clear AP pattern or show any correlation with the Bilaterian Hox code in determining axis specification8. Phylogenetic analyses of ANTP class genes have shown that the Hox and ParaHox genes are more closely related to each other than they are to the NK subclass1,7. Therefore, the current collection of genomic and phylogenetic data support the hypothesis that the NK, Hox, and ParaHox genes arose prior to the emergence of Bilaterian animals. Furthermore, it has been proposed that all three gene subclasses are derived from a hypothetical ancestral ANTP class gene that underwent extensive tandem gene duplications that ultimately created the three distinct gene clusters1. Interestingly, each of these three gene clusters has been conserved to different extents in various evolutionary lineages within Bilateria1,8. For example, vertebrates have tightly linked Hox and ParaHox clusters and disrupted NK clusters, whereas dipterans (including Drosophila) exhibit a disrupted Hox cluster but have retained a tight NK cluster1. Despite these differences, the birth and diversification of ANTP class genes have been instrumental in the evolution of the Bilaterian body plan and have contributed to the subsequent radiation of these animal taxa into nearly every ecological niche on earth.\n\nTypically, invertebrates possess a single Hox cluster, whereas vertebrates possess multiple clusters that differ among different taxa9. For example, mammalian genomes have four Hox clusters whereas teleost fishes have up to eight Hox clusters9–11. Although Hox genes and clusters are relatively well characterized in most vertebrates, the evolution of these genes within this group remains largely obscure because of the incompletely resolved phylogenetic history of these genes12. In particular, the evolutionary origins of the Hox-bearing chromosomes in mammals remain highly controversial. The classic view is that the four clusters of Hox genes in humans originated through two rounds of whole genome duplications13–15. However, over the past few years, with the rapidly increasing availability of high-quality whole genome sequence data from a variety of animal species, the evolutionary history and organization of mammalian Hox genes have been subjected to rigorous scrutiny12,16–20. Analyses of these emerging genomic datasets with advanced phylogenetic techniques have generated data that are inconsistent with the whole genome duplication hypothesis and instead favor the hypothesis that the configuration of Hox-bearing chromosomes in mammals may have resulted from small-scale events early in vertebrate evolution that include segmental duplications, independent gene duplication, and translocations12. Such advanced phylogenetic techniques will continue to prove valuable and will provide more rigorous analyses of the evolution of the Hox genes as more high-quality whole genome sequence data from more basal metazoan taxa become available.\n\n\nConservation of Hox function in antero-posterior patterning\n\nThe spatial and temporal expression patterns of Hox genes along the AP axis of flies reflect their position on the chromosome: genes at the most 3′ end are expressed earlier in development in more anterior parts of the embryo, and genes at the more 5′ position are expressed later in development in more posterior regions of the embryo9. Studies in mice have shown the spatial and temporal expression patterns of these genes are also correlated with their position from 3′ to 5′ in each cluster, indicating that the spatial and temporal collinearity of the Hox genes is conserved in mammals4–6,21. To date, Hox gene expression analyses in the vertebral column have been extended into several vertebrate taxa, including teleost fishes22, squamates4,23–25, and archosaurs4,26,27. Comparative analyses of the Hox code in several amniote taxa provide strong evidence that the evolutionary differences in the axial skeleton correspond to changes in the expression domains of Hox genes26. As more diverse taxa are sampled, the trend of deep conservation of the spatio-temporal expression and function of the Hox genes along the AP axis seems to be continually reinforced and underlies the critical roles that these genes have played in the evolution of the Bilaterian body plan.\n\nThere is an overwhelming amount of data that support that Hox genes are critical for patterning the axial skeleton in vertebrates and that changes in Hox gene expression have helped shape the evolution of novel body plans within Bilateria4,5. With these cumulative results, the origin of the snake-like body plan (as well as other snake-like squamates) with its “deregionalized” axial skeleton28,29 has been an intriguing evolutionary feature that has received considerable attention over the last decade with regard to Hox gene expression and function23,25,28–30. In limbed lizards, two distinct regional boundaries are observed in the axial skeleton: the cervical-thoracic and the thoracic-lumbar, both of which have been shown to correspond to sharp boundaries of differential Hox gene expression patterns23,25,28. In contrast, it has been reported that the snake-like body plan lacks clear boundaries, resulting in a “deregionalized” axial skeleton with an increased number of vertebra and ribs and a reduction or loss of limbs and sternum28,29. Previous studies in mice have shown that the inactivation of all three genes in the Hox10 paralogous group (Hoxa10, Hoxc10, and Hoxd10) results in the transformation of the ribless lumbar vertebrae into a posterior extension of the thorax, as defined by the presence of ectopic ribs31,32. These and many other genetic studies demonstrate that the activity of the genes in the Hox10 paralogous group controls key processes in somatic patterning that lead to the inhibition of rib development. However, expression analyses in snake embryos have shown that both Hoxa10 and Hoxc10 are expressed in rib-bearing regions of the axial skeleton, suggesting the possibility that snake Hoxa10 and Hoxc10 genes have lost the ability to suppress rib-bearing vertebrae25,30. Generation of transgenic mice that ectopically express snake Hoxa10 showed that this paralog is able to efficiently block rib formation in mice, indicating that rib-repressing properties are still present in the snake protein33. Instead, a polymorphism was identified in a Hox/Pax-responsive enhancer that is involved in Hox-mediated regulation of rib formation, which results in this enhancer being unable to respond to Hox10 proteins33. In addition, this polymorphism was also found in other animals with extended rib cages. These data indicate that the evolution of this Hox/Pax enhancer has played a critical role in the evolution of axial skeletons by modulating responses to either rib-suppressing or rib-promoting Hox genes.\n\nA recent report that more closely analyzed the morphological differences of snake vertebrae has challenged the traditional view that the anterior axial skeleton of snakes is, in fact, “deregionalized”28. Using a statistical geometric morphometric analysis on the vertebral morphology, Head and Polly28 concluded that there was no consistent difference in the shape variance between limbed and snake-like squamates and that three to four distinct vertebral regions, including the cervical, thoracic, and lumbar regions, could be identified in all taxa irrespectively of the presence or absence of limbs. In other words, snake-like body plans do indeed have regionalized precloacal axial skeletons; the differences in the morphologies of the vertebrae are just more subtle as compared with limbed reptiles. In addition, the authors asserted that the newly identified morphological boundaries of the snake vertebral columns correspond to similar mapped expression boundaries of Hox paralogs in snakes, suggesting that the AP axis of these animals is governed by a normally functioning Hox code.\n\nFrom an evolutionary perspective, the “deregionalization” of the snake axial skeleton made the assumption that this body plan evolved from an ancestor that exhibited a regionalized AP vertebral axis. The new data reported in Head and Polly challenge this assumption and instead suggest that the regionalized axial skeletons of limbed reptiles and other derived vertebrate taxa are descended from an axial plan that displayed very little regionalization in the first place28. Indeed, this hypothesis is supported by acquired fossil evidence from Paleozoic amniotes, including extinct stem members of Reptilia and Mammalia, that shows that these animals exhibited “deregionalized” axial skeletons with very subtle changes in their primaxial morphology28. These data support a model wherein regionalized vertebral columns (including the ones in snakes) are a derived feature that has arisen through modifications of a more “deregionalized” ancestral body plan. In this case, the evolution of the snake-like body plan is not an exception but rather just another example along the continuum of Hox function in sculpting derived body plans in the diverse Bilaterian taxa.\n\nIn addition to their highly conserved roles in AP patterning, numerous studies have indicated that Hox genes have been co-opted for novel functions in the development of many organ systems. For example, previous studies have shown that the expression patterns of the Hox gene Ultrabithorax (Ubx) are associated with the differential enlargement of particular hind-limb segments in different insect species34,35. In a similar fashion, the Hox gene Abdominal-B is required for the formation of the lantern organ on the posterior abdomen in the firefly36. Although studies in insects are informative, our most comprehensive understanding of co-opted Hox gene functions comes from studies in mice. These studies have identified several important roles for Hox genes in the development of organs that correspond to their expression along the AP axis. Some of the many examples include the following: the Hox3 genes in the development of the thymus, thyroid, and parathyroid37,38; Hox5 genes in lung development39,40; Hox6 genes in pancreas development41; Hox9, Hox10, and Hox11 genes in the reproductive tract42–46; Hox10 and Hox11 genes in kidney development47–49; and Hoxb13 gene for prostate development50. Although Hox genes have been shown to play important roles in many aspects of organogenesis, it has been difficult to place these highly conserved transcription factors into established regulatory networks. This represents an important gap in our understanding of Hox biology that needs to be addressed in much greater detail.\n\nIt has been well established that the diversity along the AP axis of animals results from the differential expression of Hox genes, which in turn regulate different sets of target genes that govern the formation of anatomical regions that have specific features51. However, how Hox genes encode this specificity has been a long-debated question. All Hox proteins have similar DNA-binding domains (the homeodomain) and they all bind similar DNA sequences with high affinity51–56. One well-established means by which Hox genes achieve specificity in vivo is to bind DNA co-operatively with other DNA-binding co-factors55,57. To date, the three amino acid loop extension (TALE)-homeodomain genes, which include the PBC/PBX and MEIS classes of homeodomain proteins, are the best described co-factors; however, it is clear that others exist55,57–61. The PBC/PBX class comprises fly Extradenticle (Exd) and vertebrate Pbx homeoproteins, whereas the MEIS class includes fly Homothorax (Hth) and vertebrate Meis and Prep homeoproteins57. In addition to the presence or absence of co-factors, a recent report has significantly contributed to additional understanding of how Hox genes encode specificity in Drosophila54. Briefly, these researchers identified clusters of low-affinity binding sites in enhancers of the shavenbaby (svb) gene that specifically confer binding of an Ubx-Exd complex. Mutation of these sites into high-affinity sites enabled the enhancer to respond to other Hox genes (Scr), suggesting that the native low-affinity Ubx-Exd binding sites confer specificity for Ubx-Exd dimers over other Hox proteins and probably over other homeodomain proteins as well. Interestingly, although the individual Ubx binding sites were not conserved in another fly species (Drosophila virilis), clusters of other low-affinity binding sites were identified and found to be required for enhancer function, suggesting that this mechanism may be an evolutionarily conserved strategy used by svb enhancers. Determining whether similar mechanisms convey Hox specificity in more derived Bilaterian species will be particularly informative and will provide insight into whether this mechanism is a highly conserved feature.\n\n\nFuture directions\n\nThe remarkably deep conservation of Hox gene organization and function and their profound impact on the evolution of metazoan body plans continue to fascinate evolutionary and developmental biologists today. As a result, Hox genes continue to be investigated by a large number of research groups. The focus of these studies encompasses many different aspects of Hox biology, including Hox gene regulation, identification of downstream targets, and uncovering novel functions for these proteins. In addition, Hox genes have been associated with a number of human diseases62 and this in turn supports an increased need to understand the potential role(s) of these genes in the onset and progression of disease. Finally, functional roles of Hox genes have also been identified during postnatal development63–65, and there is increasing interest in understanding the roles that these genes play in the formation of post-embryonically derived structures and the maintenance of organ systems. There remains much more to learn about Hox gene biology and thus it is certain that these genes will continue to fascinate investigators for decades to come.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHolland PW: Did homeobox gene duplications contribute to the Cambrian explosion? Zoological Lett. 2015; 1: 1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHolland PW: Evolution of homeobox genes. Wiley Interdiscip Rev Dev Biol. 2013; 2(1): 31–45. PubMed Abstract | Publisher Full Text\n\nLewis EB: A gene complex controlling segmentation in Drosophila. Nature. 1978; 276(5688): 565–70. PubMed Abstract | Publisher Full Text\n\nBurke AC, Nelson CE, Morgan BA, et al.: Hox genes and the evolution of vertebrate axial morphology. Development. 1995; 121(2): 333–46. PubMed Abstract\n\nMallo M, Wellik DM, Deschamps J: Hox genes and regional patterning of the vertebrate body plan. Dev Biol. 2010; 344(1): 7–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWellik DM: Hox patterning of the vertebrate axial skeleton. Dev Dyn. 2007; 236(9): 2454–63. PubMed Abstract | Publisher Full Text\n\nLarroux C, Fahey B, Degnan SM, et al.: The NK homeobox gene cluster predates the origin of Hox genes. Curr Biol. 2007; 17(8): 706–10. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nReddy PC, Unni MK, Gungi A, et al.: Evolution of Hox-like genes in Cnidaria: Study of Hydra Hox repertoire reveals tailor-made Hox-code for Cnidarians. Mech Dev. 2015; 138(Pt 2): 87–96. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPascual-Anaya J, D'Aniello S, Kuratani S, et al.: Evolution of Hox gene clusters in deuterostomes. BMC Dev Biol. 2013; 13: 26. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHoegg S, Meyer A: Hox clusters as models for vertebrate genome evolution. Trends Genet. 2005; 21(8): 421–4. PubMed Abstract | Publisher Full Text\n\nKuraku S, Meyer A: The evolution and maintenance of hox gene clusters in vertebrates and the teleost-specific genome duplication. Int J Dev Biol. 2009; 53(5–6): 765–73. PubMed Abstract | Publisher Full Text\n\nAbbasi AA: Diversification of four human Hox gene clusters by step-wise evolution rather than ancient whole-genome duplications. Dev Genes Evol. 2015; 225(6): 353–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKasahara M: The 2R hypothesis: an update. Curr Opin Immunol. 2007; 19(5): 547–52. PubMed Abstract | Publisher Full Text\n\nOhno S: Evolution by Gene Duplication. Berlin, Heidelberg: Springer Berlin Heidelberg, 1970. Publisher Full Text\n\nPutnam NH, Butts T, Ferrier DE, et al.: The amphioxus genome and the evolution of the chordate karyotype. Nature. 2008; 453(7198): 1064–71. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAbbasi AA: Unraveling ancient segmental duplication events in human genome by phylogenetic analysis of multigene families residing on Hox-cluster paralogons. Mol Phylogenet Evol. 2010; 57(2): 836–48. PubMed Abstract | Publisher Full Text\n\nAbbasi AA, Grzeschik KH: An insight into the phylogenetic history of Hox linked gene families in vertebrates. BMC Evol Biol. 2007; 7: 239. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmbreen S, Khalil F, Abbasi AA: Integrating large-scale phylogenetic datasets to dissect the ancient evolutionary history of vertebrate genome. Mol Phylogenet Evol. 2014; 78: 1–13. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAsrar Z, Haq F, Abbasi AA: Fourfold paralogy regions on human Hox-bearing chromosomes: role of ancient segmental duplications in the evolution of vertebrate genome. Mol Phylogenet Evol. 2013; 66(3): 737–47. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHughes AL, da Silva J, Friedman R: Ancient genome duplications did not structure the human Hox-bearing chromosomes. Genome Res. 2001; 11(5): 771–80. PubMed Abstract | Free Full Text\n\nDuboule D, Dollé P: The structural and functional organization of the murine Hox gene family resembles that of Drosophila homeotic genes. EMBO J. 1989; 8(5): 1497–505. PubMed Abstract | Free Full Text\n\nMorin-Kensicki EM, Melancon E, Eisen JS: Segmental relationship between somites and vertebral column in zebrafish. Development. 2002; 129(16): 3851–60. PubMed Abstract | F1000 Recommendation\n\nCohn MJ, Tickle C: Developmental basis of limblessness and axial patterning in snakes. Nature. 1999; 399(6735): 474–9. PubMed Abstract | Publisher Full Text\n\nOhya YK, Kuraku S, Kuratani S: Hox code in embryos of Chinese soft-shelled turtle Pelodiscus sinensis correlates with the evolutionary innovation in the turtle. J Exp Zool B Mol Dev Evol. 2005; 304(2): 107–18. PubMed Abstract | Publisher Full Text\n\nWoltering JM, Vonk FJ, Müller H, et al.: Axial patterning in snakes and caecilians: evidence for an alternative interpretation of the Hox code. Dev Biol. 2009; 332(1): 82–9. PubMed Abstract | Publisher Full Text\n\nBöhmer C, Rauhut OW, Wörheide G: New insights into the vertebral Hox code of archosaurs. Evol Dev. 2015; 17(5): 258–69. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMansfield JH, Abzhanov A: Hox expression in the American alligator and evolution of archosaurian axial patterning. J Exp Zool B Mol Dev Evol. 2010; 314(8): 629–44. PubMed Abstract | Publisher Full Text\n\nHead JJ, Polly PD: Evolution of the snake body form reveals homoplasy in amniote Hox gene function. Nature. 2015; 520(7545): 86–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWoltering JM: From lizard to snake; behind the evolution of an extreme body plan. Curr Genomics. 2012; 13(4): 289–99. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDi-Poï N, Montoya-Burgos JI, Miller H, et al.: Changes in Hox genes' structure and function during the evolution of the squamate body plan. Nature. 2010; 464(7285): 99–103. PubMed Abstract | Publisher Full Text\n\nCarapuço M, Nóvoa A, Bobola N, et al.: Hox genes specify vertebral types in the presomitic mesoderm. Genes Dev. 2005; 19(18): 2116–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWellik DM, Capecchi MR: Hox10 and Hox11 genes are required to globally pattern the mammalian skeleton. Science. 2003; 301(5631): 363–7. PubMed Abstract | Publisher Full Text\n\nGuerreiro I, Nunes A, Woltering JM, et al.: Role of a polymorphism in a Hox/Pax-responsive enhancer in the evolution of the vertebrate spine. Proc Natl Acad Sci U S A. 2013; 110(26): 10682–6. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMahfooz N, Turchyn N, Mihajlovic M, et al.: Ubx regulates differential enlargement and diversification of insect hind legs. PLoS One. 2007; 2(9): e866. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMahfooz NS, Li H, Popadić A: Differential expression patterns of the Hox gene are associated with differential growth of insect hind legs. Proc Natl Acad Sci U S A. 2004; 101(14): 4877–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStansbury MS, Moczek AP: The function of Hox and appendage-patterning genes in the development of an evolutionary novelty, the Photuris firefly lantern. Proc Biol Sci. 2014; 281(1782): 20133333. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDollé P, Izpisúa-Belmonte JC, Brown JM, et al.: HOX-4 genes and the morphogenesis of mammalian genitalia. Genes Dev. 1991; 5(10): 1767–7. PubMed Abstract | Publisher Full Text\n\nManley NR, Capecchi MR: Hox group 3 paralogs regulate the development and migration of the thymus, thyroid, and parathyroid glands. Dev Biol. 1998; 195(1): 1–15. PubMed Abstract | Publisher Full Text\n\nBoucherat O, Montaron S, Bérubé-Simard FA, et al.: Partial functional redundancy between Hoxa5 and Hoxb5 paralog genes during lung morphogenesis. Am J Physiol Lung Cell Mol Physiol. 2013; 304(12): L817–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHrycaj SM, Dye BR, Baker NC, et al.: Hox5 Genes Regulate the Wnt2/2b-Bmp4-Signaling Axis during Lung Development. Cell Rep. 2015; 12(6): 903–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLarsen BM, Hrycaj SM, Newman M, et al.: Mesenchymal Hox6 function is required for mouse pancreatic endocrine cell differentiation. Development. 2015; 142(22): 3859–68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBenson GV, Lim H, Paria BC, et al.: Mechanisms of reduced fertility in Hoxa-10 mutant mice: uterine homeosis and loss of maternal Hoxa-10 expression. Development. 1996; 122(9): 2687–96. PubMed Abstract\n\nGendron RL, Paradis H, Hsieh-Li HM, et al.: Abnormal uterine stromal and glandular function associated with maternal reproductive defects in Hoxa-11 null mice. Biol Reprod. 1997; 56(5): 1097–105. PubMed Abstract | Publisher Full Text\n\nPodlasek CA, Seo RM, Clemens JQ, et al.: Hoxa-10 deficient male mice exhibit abnormal development of the accessory sex organs. Dev Dyn. 1999; 214(1): 1–12. PubMed Abstract | Publisher Full Text\n\nRaines AM, Adam M, Magella B, et al.: Recombineering-based dissection of flanking and paralogous Hox gene functions in mouse reproductive tracts. Development. 2013; 140(14): 2942–52. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nTaylor HS, Vanden Heuvel GB, Igarashi P: A conserved Hox axis in the mouse and human female reproductive system: late establishment and persistent adult expression of the Hoxa cluster genes. Biol Reprod. 1997; 57(6): 1338–45. PubMed Abstract | Publisher Full Text\n\nSchwab K, Hartman HA, Liang HC, et al.: Comprehensive microarray analysis of Hoxa11/Hoxd11 mutant kidney development. Dev Biol. 2006; 293(2): 540–54. PubMed Abstract | Publisher Full Text\n\nWellik DM, Hawkes PJ, Capecchi MR: Hox11 paralogous genes are essential for metanephric kidney induction. Genes Dev. 2002; 16(11): 1423–32. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nYallowitz AR, Hrycaj SM, Short KM, et al.: Hox10 genes function in kidney development in the differentiation and integration of the cortical stroma. PLoS One. 2011; 6(8): e23410. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEconomides KD, Capecchi MR: Hoxb13 is required for normal differentiation and secretory function of the ventral prostate. Development. 2003; 130(10): 2061–9. PubMed Abstract | Publisher Full Text\n\nMcGinnis W, Krumlauf R: Homeobox genes and axial patterning. Cell. 1992; 68(2): 283–302. PubMed Abstract | Publisher Full Text\n\nAkam M: Hox and HOM: homologous gene clusters in insects and vertebrates. Cell. 1989; 57(3): 347–9. PubMed Abstract | Publisher Full Text\n\nBerger MF, Badis G, Gehrke AR, et al.: Variation in homeodomain DNA binding revealed by high-resolution analysis of sequence preferences. Cell. 2008; 133(7): 1266–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCrocker J, Abe N, Rinaldi L, et al.: Low affinity binding site clusters confer hox specificity and regulatory robustness. Cell. 2015; 160(1–2): 191–203. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMann RS, Lelli KM, Joshi R: Hox specificity unique roles for cofactors and collaborators. Curr Top Dev Biol. 2009; 88: 63–101. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNoyes MB, Christensen RG, Wakabayashi A, et al.: Analysis of homeodomain specificities allows the family-wide prediction of preferred recognition sites. Cell. 2008; 133(7): 1277–89. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMoens CB, Selleri L: Hox cofactors in vertebrate development. Dev Biol. 2006; 291(2): 193–206. PubMed Abstract | Publisher Full Text\n\nRezsohazy R, Saurin AJ, Maurel-Zaffran C, et al.: Cellular and molecular insights into Hox protein action. Development. 2015; 142(7): 1212–27. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAmin S, Donaldson IJ, Zannino DA, et al.: Hoxa2 selectively enhances Meis binding to change a branchial arch ground state. Dev Cell. 2015; 32(3): 265–77. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nGong KQ, Yallowitz AR, Sun H, et al.: A Hox-Eya-Pax complex regulates early kidney developmental gene expression. Mol Cell Biol. 2007; 27(21): 7661–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYallowitz AR, Gong KQ, Swinehart IT, et al.: Non-homeodomain regions of Hox proteins mediate activation versus repression of Six2 via a single enhancer site in vivo. Dev Biol. 2009; 335(1): 156–65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQuinonez SC, Innis JW: Human HOX gene disorders. Mol Genet Metab. 2014; 111(1): 4–15. PubMed Abstract | Publisher Full Text\n\nMandeville I, Aubin J, LeBlanc M, et al.: Impact of the loss of Hoxa5 function on lung alveogenesis. Am J Pathol. 2006; 169(4): 1312–27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPineault KM, Swinehart IT, Garthus KN, et al.: Hox11 genes regulate postnatal longitudinal bone growth and growth plate proliferation. Biol Open. 2015; 4(11): 1538–48. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorgan R: Hox genes: a continuation of embryonic patterning? Trends Genet. 2006; 22(2): 67–9. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13733",
"date": "10 May 2016",
"name": "Yacine Graba",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13734",
"date": "10 May 2016",
"name": "Moises Mallo",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13732",
"date": "10 May 2016",
"name": "Robb Krumlauf",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-859
|
https://f1000research.com/articles/4-1450/v1
|
15 Dec 15
|
{
"type": "Opinion Article",
"title": "The TOUCH program and natalizumab: Fundamental flaw in patient protection",
"authors": [
"Jagannadha Avasarala"
],
"abstract": "Many drugs have been approved by the Food and Drug Administration (FDA) since 1993 for treatment of relapsing forms of multiple sclerosis (MS). One such drug is natalizumab (Tysabri, Biogen Idec and Elan pharmaceuticals) which has enjoyed great success in the management of MS since its re-introduction in 2006. One of the complications of using natalizumab is the risk of development of progressive multifocal leukoencephalopathy (PML). To mitigate the risk of PML development, Biogen Idec initiated the TOUCH program – this strategy helps monitor the disease. Clinical vigilance is remains key in the early diagnosis of PML but serological testing for the John Cunningham Virus Antibody (JCV) helps with risk stratification of PML. However, some physicians do not test for the JCV Ab and since they are not required to send such data to the company or inform the patient, one red flag for suspicion of PML is lost particularly if the patient is asymptomatic. This undercuts the premise of the TOUCH program. In an ideal world, reporting JCV Ab status should be made mandatory since that ensures a basic tenet of the program is met – to identify patients at increased risk of developing PML and make appropriate recommendations based on that finding. Lack of requirement of reporting of this vital finding opens the door for uncertainty in assessment of risk PML development and everyone remains in the dark till it may be too late. This is unacceptable when the company created the TOUCH program specifically with intent to track PML risk in patients on natalizumab. It makes no scientific sense to let the drug be used without setting stringent criteria given the possibility of PML development.",
"keywords": [
"natalizumab",
"multiple sclerosis",
"progressive multifocal leukoencephalopathy",
"TOUCH",
"John Cunningham Virus antibody",
"JCV Ab"
],
"content": "\n\nNatalizumab is the first monoclonal antibody approved for the treatment of relapsing forms of multiple sclerosis (MS) and is used in more than 50 countries. Natalizumab is a recombinant humanized monoclonal IgG4 antibody that binds to alpha 4 beta 1 integrin and interferes with alpha 4-mediated binding to extracellular matrix and endothelial lining, vascular cell adhesion molecule (VCAM1) and fibronectin. After its initial approval in 2004 by the FDA, it was voluntarily withdrawn in early 2005 after two patients with MS in the SENTINEL trial and 1 patient with Crohn’s disease were diagnosed with progressive multifocal leukoencephalopathy (PML)1–3.\n\nThe drug was reapproved in 2006 and recommendations were made in the US to limit its use to highly active relapsing-remitting MS (with more than two relapses per year) and to those patients who did not respond or tolerate first-line treatment such as interferon beta-1a, interferon beta-1b, or glatiramer acetate. As well, a restricted risk minimization plan was also initiated to better assess an individual’s risk of PML: Tysabri Outreach: Unified Commitment to Health [TOUCH]. This created a system where only prescribers and patients enrolled in the TOUCH program could prescribe and receive the drug. Additionally, only certain pharmacies and infusion sites authorized by the TOUCH prescribing program could dispense and infuse natalizumab. The primary goals of the program were to inform prescribers, infusion center healthcare providers and particularly patients, about the risk of development of PML associated with natalizumab use including the positive association of increased risk of PML and a) treatment duration, b) prior immunosuppressant use and c) JCV Ab status. The TOUCH program also includes information on and warnings against concurrent use of natalizumab with antineoplastic, immunosuppressant, or immunomodulating agents and in patients who are immunocompromised. The TOUCH program is solely designed to facilitate PML risk assessment at the individual patient level and promote early diagnosis and timely discontinuation of natalizumab in the event of suspected PML.\n\nHowever, the way the TOUCH program is applied in the real world is less than desirable. For instance, the FDA has not approved the validity or applicability of the JCV Ab index (anti-JCV Ab levels in serum/plasma) which may differentiate PML risk in JCV-Ab positive MS patients with no prior immunosuppressant use4. Despite its lack of FDA approval status, the JCV Ab index is widely used by MS clinicians in the risk evaluation of PML development. Clinicians worry once the index begins to rise although doubling the index value does not automatically confer twice the risk of PML development. Since the index is not FDA-approved, the TOUCH program cannot mandate its routine use but every patient who has some basic understanding of the PML saga in MS wants to know his/her JCV Ab index. Laboratories run the test, clinicians use it for better or worse and yet the TOUCH program cannot adopt it. It is not an inherent flaw of the TOUCH program itself but sooner rather than later, the FDA should establish whether the JCV Ab index is valid and whether it can be part of a modified TOUCH program or not.\n\nAnother confusing test that some clinicians continue to use without rhyme or reason and on a monthly basis is the measurement of JCV DNA viremia5. This too, akin to the JCV Ab index, is not part of the TOUCH program risk assessment strategy for PML. Although viremia by itself is not a predictor of PML risk, that it can occur in JCA Ab negative patients ‘raises other issues’ according to authors who advocate ‘periodic monitoring’ over the course of the treatment with natalizumab without offering specific time-specific testing protocols5. Again, the TOUCH program administrators cannot be responsible if testing for JCV viremia does not have scientific relevance and if uninformed clinicians continue to pursue JCV DNA studies religiously, falsely assuming that they are tracking PML – they are not. The test is superfluous and literally a waste of patient’s blood and money.\n\nThe biggest fundamental flaw in the TOUCH program is Biogen’s reauthorization questionnaire wherein physicians are allowed to prescribe natalizumab despite the fact that JCV Ab status is not tested or necessarily even reported. Therefore, a clinician can order natalizumab to be administered to patients without periodically reporting (every 6 months) the patient’s JCV Ab status to the company as this is not mandatory and without it, patients can still stay in the TOUCH program. Most clinicians do track PML using JCV Ab status every 6 months as required but as a neurologist and a fellowship-trained multiple sclerosis physician, I have seen patients without JCV Ab testing or reporting who yet continue to be in the TOUCH program. It is also true that JCV Ab status, if positive, does not imply PML development, but it begs the question as to why the TOUCH program does not insist that JCV Ab status be reported every 6 months. A simple solution would be to make the JCV Ab status available to the company and if the patient and their physician decide to continue the drug despite JCV Ab status being positive, that is a choice between the patient and physician. Obviously, JCV Ab positive status is one of many factors that can increase the risk of PML development – use of the drug beyond two years and prior immunosuppressant use also increase the risk of PML. Clinicians understand and agree that early diagnosis of PML hinges on clinical vigilance.\n\nSince Biogen Idec and the FDA are interested in halting PML in its tracks, and there have been, as of September 4, 2015, a total of 588 confirmed cases of PML while on natalizumab6, it must be obvious for all those concerned with patient safety that it is necessary to plug this loophole. Strangely enough, confirmed PML cases from natalizumab use are not available in a database for researchers to probe into individual (personal details can be encrypted) cases for analysis. The primary goal of the TOUCH program is to address risk stratification of PML and therefore, allowing clinicians to continue to prescribe natalizumab without knowledge of the JCV Ab status is a huge risk. It would be an easy recommendation to make JCV Ab testing mandatory; making JCV Ab status reporting the sine qua non for prescribing this drug adds one more layer of protection to patients.\n\nIt is unknown if any of the 588 reported cases of PML fall into the category that I have described – even if only one patient did, this would call into question whether it was preventable and what the role of the TOUCH program should be in preventing it. One wonders what proportion of patients do not have their JCV Ab status reported across the globe while in the TOUCH program. Since hundreds of PML cases are already known, and more will likely continue to be reported, it is conceivable that questions will be raised as to whether more could have been done to prevent such cases. I hope there are no instances of PML owing to omission of JCV Ab status evaluation but I also think it is time for FDA to act now to prevent future lapses and avoid legal nightmares. My suggestion would be to make reporting of JCV Ab status mandatory for all patients on natalizumab in the TOUCH program - from a pharmacovigilance perspective, this makes perfect sense.",
"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\nReferences\n\nKleinschmidt-DeMasters BK, Tyler KL: Progressive multifocal leukoencephalopathy complicating treatment with natalizumab and interferon beta-1a for multiple sclerosis. N Engl J Med. 2005; 353(4): 369–374. PubMed Abstract | Publisher Full Text\n\nLanger-Gould A, Atlas SW, Green AJ, et al.: Progressive multifocal leukoencephalopathy in a patient treated with natalizumab. N Engl J Med. 2005; 353(4): 375–381. PubMed Abstract | Publisher Full Text\n\nVan Assche G, Van Ranst M, Sciot R, et al.: Progressive multifocal leukoencephalopathy after natalizumab therapy for Crohn’s disease. N Engl J Med. 2005; 353(4): 362–368. PubMed Abstract | Publisher Full Text\n\nPlavina T, Subramanyam M, Bloomgren G, et al.: Anti-JC virus antibody levels in serum or plasma further define risk of natalizumab-associated progressive multifocal leukoencephalopathy. Ann Neurol. 2014; 76(6): 802–812. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMajor EO, Frohman E, Douek D: JC viremia in natalizumab-treated patients with multiple sclerosis. N Engl J Med. 2013; 368(23): 2240–2241. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQuarterly safety update, Biogen Idec."
}
|
[
{
"id": "11563",
"date": "03 Feb 2016",
"name": "David B Clifford",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think that advocating to legislate science that is admittedly not yet accepted as useful is not such a good idea. Changing the goal to driving data collection to decide risk stratification that avoids PML might be a good idea. TOUCH was not created to provide risk stratification, but to help assure early diagnosis. If there is any enhancement, it might be better to advocate for frequent MRI which do appear to improve outcomes for PML. To date, there is no prospective evidence that antibody monitoring prevents PML, and indeed if anything the evidence is that it does not, since cases continue while it is available. I would suggest re-working the recommendation to a program that helps prove if antibody data actually can help physicians prevent PML. I recommended removing the distracting paragraph about JC DNA.I would recommend including frequency of imaging as part of TOUCH since it appears to help make earlier diagnosis and improve outcomes. I think questioning whether TOUCH is effective at present is realistic.",
"responses": [
{
"c_id": "1794",
"date": "04 Feb 2016",
"name": "Jagannadha Avasarala",
"role": "Author Response",
"response": "I thank Dr Clifford for his erudite observations. 1, I agree that the TOUCH program helps in early diagnosis - in fact, the following are explicitly stated on the touchprogram.com, thus: Inform prescribers, infusion center healthcare providers, and patients about the risk of progressive multifocal leukoencephalopathy (PML) associated with TYSABRI including the increased risk of PML with treatment duration and prior immunosuppressant use.Warn against concurrent use with antineoplastic, immunosuppressant, or immunomodulating agents and in patients who are immunocompromised.Promote early diagnosis of PML and timely discontinuation of TYSABRI in the event of suspectedFurthermore, in the important safety information section, it is clearly noted that risk factors for the development of PML include duration of therapy, prior use of immunosuppressants, and presence of anti-JCV antibodies.As clinicians, we all know that 'early diagnosis' of PML includes the STRATIFY testing protocol approved by the FDA in 2012 and that test is designed specifically to assess PML risk. We also understand and know that JCV Ab negative status also carries risk of PML development but what we cannot ignore what the safety information (noted above) and STRATIFY testing are designed to do. Therefore, uncoupling the TOUCH program from testing/reporting JCV Ab status is not only dangerous and fallacious, it runs counter to the argument that there is any 'risk stratification' being done if this simple testing is forgotten or discarded. No one can claim that JCV Ab monitoring prevents PML and nor do I state that; but assessing PML risk with STRATIFY is a fundamental principle of the test or we could discard the test altogether ! That frequent MRI testing, clinical surviellance (most critical) and patient self-reporting of new symptoms or worsening of existing symptoms is paramount to the diagnosis of PML is a well established fact, based on scientific evidence.If JCV Ab testing is of such low importance, why does the TOUCH program questionnaire include this as part of their questionnaire ? One cannot have it both ways. Either the testing is critical or we do not test it at all and shun the JCV Ab testing as well as the JCV index. Why have a test approved by the FDA (Stratify), create the TOUCH program to monitor and track PML, include JCV Ab status in the questionnaire that is generated by the company and yet reject the very idea of monitoring for PML by throwing away the JCV Ab testing ? The statement that \"TOUCH program was created to help assure early diagnosis\" utterly does not hold water if JCV Ab testing is not done. As pointed out in my paper, the questionnaire itself includes it ! What is the inclusion for ? It is not exactly for statistical purposes, is it ?Dr Avasarala, MD, PhD"
},
{
"c_id": "1795",
"date": "04 Feb 2016",
"name": "Jagannadha Avasarala",
"role": "Author Response",
"response": "I would also submit the following, additional comments.The TOUCH program, when it was first introduced, did not have the benefit of STRATIFY, approved in 2012. But once JCV Ab testing was FDA approved in 2012, and JCV Ab status testing was part of the TOUCH questionnaire to continue Tysabri use, it became an essential tool to monitor PML risk (in fact, the word Stratify is itself a connotation to categorize risk of PML) so how it is part of a strategy to assess PML risk and yet can be ignored at the same time does not add up. If patients are to be protected or their risk explained to them, every single tool available needs to be put to use. Simple as that."
}
]
},
{
"id": "12282",
"date": "09 Feb 2016",
"name": "Nicholas Schwab",
"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 first point I would like to make (as it is mentioned in the abstract) is that TOUCH has not been introduced by Biogen as a method to mitigate PML risk, but as a method to inform patients and physicians of the PML risk and to monitor the patients for early signs of PML allowing for a better diagnosis and treatment. While some might argue that PML prevention should be the ultimate goal of TOUCH, it has not been designed to do that and one can hardly blame the program for not reaching a goal it has not been set up to achieve.The FDA approval of the JCV index should indeed be pursued, as the author is right in assuming that patients and physicians already use the JCV index in risk stratification decisions and the sooner the FDA rules on the biomarker, the better, so it can be applied during TOUCH in a coordinated and sensible way.The data concerning JCV viremia and PML risk does not support it as a risk biomarker and I would either downright state that or remove the paragraph.I agree with the author that the consequent monitoring and application of the JCV serology would be a step towards reducing PML incidence, as JCV serology is still the most sensitive biomarker with regard to PML development. However, I would personally say that it is up to patient and physician to either use the serology or choose not to. While the goal of maximum safety is a commendable one, I would argue that personal choice on whether a patient wants to know their JCV status is even more important. It would be a different situation, if the JCV serology had a high specificity, then the use of natalizumab should be restricted to anti-JCV negative patients. With a low specificity of ca. 45% it can be reasoned that a patient does not want to know their status, if they urgently need to use natalizumab anyway and prefer not be worried about their PML risk.While it would be a great data resource to know and monitor the JCV serostatus (and potentially index) of all TOUCH patients, to force a biomarker with low specificity on patients, who might choose not to use it, would have far-reaching consequences. The knowledge of their JCV serostatus has not prevented the occurrence of the 300+ PML, where it was available before, so I do not think that the mandatory use would help in this regard. The biomarker should, however, be available to all patients, who want to use it, so no PML cases develop, where the patient was unaware of their possibility to test for anti-JCV antibodies. To my knowledge, this is already the case. The TOUCH program should be updated in the future to include possible alternative biomarkers as well and serve as a monitoring platform.Having said that, I fully support the author’s with for a usable database, where physicians and researcher can access the data of the PML patients for research purposes to get a better handle on this devastating disease.",
"responses": [
{
"c_id": "1806",
"date": "10 Feb 2016",
"name": "Jagannadha Avasarala",
"role": "Author Response",
"response": "I thank Dr Schwab for his insight and comments on my article. Here are my responses, itemized.1. To quote the TOUCH program official website statement verbatim, under the sub-heading of 'a commitment to patient safety', the following is noted - Because of the risk of PML, TYSABRI® (natalizumab) is available only through a restricted program under a Risk Evaluation and Mitigation Strategy (REMS) called the TOUCH® Prescribing Program.2. One has to also note that REMS, an FDA mandated program, noted the following for TYSABRI use - To inform prescribers, infusion center healthcare providers, and patients about the risk of progressive multifocal leukoencephalopathy (PML) associated with TYSABRI including the a) increased risk of PML with longer treatment duration, b) prior immunosuppressant use and c) the presence of anti-JCV antibodies.The above mentioned strategies came to a head with the development of STRATIFY, a test developed to stratify PML risk, and approved by the FDA in 2012 to monitor PML risk. As well, Biogen has a questionnaire that all healthcare providers enrolled in the TOUCH program have to complete every 6 months that includes a section regarding the JCV Ab status. My question is simple - what is the point of the TOUCH program, STRATIFY test, FDA approval of risk mitigation strategies, inclusion of the JCV Ab status in the questionnaire, etc., if physicians are allowed to discard the very test that is supposed to protect a patient by stratification of risk as defined by the guidelines ? Clinical surveillance, frequent MRI evaluations, history of use of other immuno-suppressant drugs in the past, as well as duration are all factors that drive PML risk higher but what of the company that put all the pieces of risk evaluation in the first place ? One cannot walk away from the basic tenet in this discussion with semantics - patient protection from PML in TYSABRI users. From 2012 and beyond, after the STRATIFY was developed, there is no excuse for Biogen to let physicians prescribe TYSABRI without checking for JCV Ab status and certainly one way of reassuring the medical community would be to a) make testing mandatory for TYSABRI continuation and b) make the PML database open to researchers to investigate if cases were indeed missed as a result of this simple error. Jagannadha Avasarala"
}
]
}
] | 1
|
https://f1000research.com/articles/4-1450
|
https://f1000research.com/articles/5-853/v1
|
09 May 16
|
{
"type": "Review",
"title": "Can free open access resources strengthen knowledge-based emerging public health priorities, policies and programs in Africa?",
"authors": [
"Ernest Tambo",
"Ghislaine Madjou",
"Christopher Khayeka-Wandabwa",
"Emmanuel N. Tekwu",
"Oluwasogo A. Olalubi",
"Nicolas Midzi",
"Louis Bengyella",
"Ahmed A. Adedeji",
"Jeanne Y. Ngogang",
"Ghislaine Madjou",
"Christopher Khayeka-Wandabwa",
"Emmanuel N. Tekwu",
"Oluwasogo A. Olalubi",
"Nicolas Midzi",
"Louis Bengyella",
"Ahmed A. Adedeji",
"Jeanne Y. Ngogang"
],
"abstract": "Tackling emerging epidemics and infectious diseases burden in Africa requires increasing unrestricted open access and free use or reuse of regional and global policies reforms as well as timely communication capabilities and strategies. Promoting, scaling up data and information sharing between African researchers and international partners are of vital importance in accelerating open access at no cost. Free Open Access (FOA) health data and information acceptability, uptake tactics and sustainable mechanisms are urgently needed. These are critical in establishing real time and effective knowledge or evidence-based translation, proven and validated approaches, strategies and tools to strengthen and revamp health systems. As such, early and timely access to needed emerging public health information is meant to be instrumental and valuable for policy-makers, implementers, care providers, researchers, health-related institutions and stakeholders including populations when guiding health financing, and planning contextual programs.",
"keywords": [
"Open access",
"free access",
"information",
"policy",
"evidence",
"program",
"intervention",
"Africa"
],
"content": "Introduction\n\nIn recent times, the persistence and unprecedented emergence of rising epidemics and infectious diseases in Africa and worldwide triggered numerous public health declarations of international concern1. These local and global uncertainties and potential consequences have prompted questions and reflections on the usefulness and implications of unlocked and unprocessed available massive database resources from different national, regional and international funded projects overtime in low and middle income countries (LMICs) and particularly in Africa. Can free open access (FOA) to these valuable resources improve evidence-based decision making policies, health planning and adequate funding allocation, innovative programs and strategic interventions performance and effectiveness to most vulnerable populations’ health and socio-economic benefits? Emerging and re-emerging infectious diseases epidemics are rampant, ranging from Ebola, influenza, Lassa fever, HIV SARS and MERS-CoV, Zika to other zoonotic diseases existing as potential threats with sporadic epidemics in old and new regions2,3.\n\nThere is a need for availability of data and information on human-vector-pathogen-ecosystem interfaces, drugs and vaccines development as well as diagnostics techniques and tools from preclinical to clinical levels. It is critical that the information is used in an equitable, ethical and transparent manner. Operational research projects in libraries, national archiving, journals, local and international1,4 scholarly institutions and centers are partially or yet to be fully tapped into maximizing and ensuring improvements of health and disease information, knowledge and empowerment for all generations5.\n\nPrevious literature reviews have shown that open access data and information are of great importance and valuable assets in information sharing, education exchanges and capacity development. This FOA necessity has practically been laid bare by the recent from West Africa Ebola to Zika epidemics crisis where experts across fields including clinical' neonatal and pediatrics have been challenged. Henceforth, academic journals, libraries sources, local and internal Non-governmental organizations (NGOs) data, data from research funded or non-funded projects, centers and institutions should be committed to FOA data and results sharing relevant to the current Zika public health crisis and future emergencies for rapidly emergency mobilization and response. Moreover, the approach has proved to be useful in translation and application of proven and reliable knowledge in guiding effective decision making policies, lifestyle adaptations and contextual programs and strategies in improving public health social economic development and well-being of local and global community2,4,6,7. Most data and information often used in global policies and initiatives are either guaranteed as free by the World Health Organization (WHO) and partners philanthropic organizations, whereas the bulk of support references and documents are not readily accessible to most African scholars, but mainly to policy-makers and implementers8. Equally, limiting access to younger generations of researchers and students who cannot afford the fee to access publication in high impact placed journals, provide highly condensed information not easily informative to those in much need.\n\nThe free open access core concept can be characterized by removal of price barriers, no subscription fees and permission barriers, no copyright and licensing restrictions to royalty free literature, to make data and information available to all populations2,8. “Fee free open access to health data and information for all generations offers a new public health paradigm shift and opportunities to meet the knowledge, lessons learnt and experiences gaps and needs in Africa. Advocacy and mitigation on lack to limited access of existing and emerging data, and information sharing is necessary in embracing regional and global open access. These novelties in information sharing approaches towards collective learning and participative engagement for sound knowledge and empowerment for better health, information exchange for equity in quality education and utilization are paramount for human and societal benefits. It is of fundamental importance to increase multi-disciplinary and inter-sectoral partnerships and collaborations not only to understand and fill the gaps through joint or independent research, but also to be able to use and mine unrestricted data and information for public health good, economic growth and sustainable development9,10. Although decades of funded and non-funded programs and projects in both developed and developing countries have generated millions of publications and databases on emerging and infectious diseases of poverty2,4,6. The impact of policy-translation of lessons learnt and experiences gained are seldom and limited in applications mostly in developing countries. As most LMICs are still challenged with weak health systems and low literacy mainly in remote rural areas and areas of political instability, inadequacies in health funding allocation and resources capacity, poor accountability and governance are present. Moreover, inefficiencies in management and lack of a multi-sectoral approach to access and use local or national data repositories in a structured manner prevent both mainstream national and regional economic development11. Furthermore, the usefulness in forecasting, prevention and management or smart response of emergency situations and disasters are yet to be fully documented and demonstrated in Africa. FOA viability and benefit in most tropical endemics and epidemics-prone developed and developing countries affirmed that the vast majority of metadata and database platforms are still locked (inaccessible and unavailable) for public use and untapped to global community multi-dimensional gains.\n\nIncreasing unrestricted and FOA use or reuse as well as timely reporting or communication capabilities strategies are urgently needed in promoting and scaling up data and information sharing and exchange between African researchers, partners and collaborators1,3,12,13. The strength of scaling FOA in developing countries will entail but not limited to: 1) increasing real time and effective knowledge- or evidence-based translation of proven and validated approaches, 2) strategies and tools in strengthening health systems and revamping early and timely access to much needed information by policy-makers, and 3) enhanced guided health financing and capacity development by health institutions and related stakeholders, and strengthening contextual programs and activities planning, transparency and accountability.\n\nThis paper assesses the values and benefits of open, free of charge data and information access and availability in strengthening health systems policies, financing, promoting knowledge-based programs and targeted interventions directed to forecast, prevent, reduce and/or manage the growing emerging threats and epidemics as well as infectious diseases of poverty in LMICs, especially in Africa.\n\n\nFOA resources and public health needs and demands in LMICs\n\nThe growing burden of emerging epidemics and infectious diseases have been documented in demoting health systems in rural and urban settings in Africa. It is important to assess and understand why and how open data and information access is needed in the context of health and diseases epidemics. Also, what capacity development and training are needed to translate these various valuable datasets and database assets if freely available into knowledge-based innovations needed to revolutionize Africa and global health capabilities, and opportunities to prevent and control emerging epidemics and infectious diseases of poverty1,3,13,14.\n\nThe current trends of globalization of trade and travel, intense urbanization, economic slowdown are coupled with rising of double epidemics burden (emerging infectious diseases and chronic diseases). Thus, there is an urgent need for open data and information access promotion, advocacy and awareness. This is critical in strengthening and improving the strategic value and usefulness of knowledge-based innovations, teaching and learning, key sources and assets of policy transformation oriented research and primary care innovations (e.g., routine to universal immunization, essential medicines and nutrition). Adopting and adapting open access proven lessons learnt and experiences to alleviate sufferings and poverty, health literacy access and delivery inequities amongst vulnerable populations in Africa is very important2,5,15,16. The evolving use of electronic data and digital delivery platforms to support open access interactive literacy, communication and empowerment of health is a vital need in increasing care acceptability, uptake and scaling up positive cultural ad behavioral changes relevant for communicable and non-communicable diseases vigilance and resilience1,17,18. However, with restricted content access, such anticipated evolution in terms of accurate timing and relevant knowledge among experts remains to be a blatant wish as technology and information are not mutually exclusive. Digital technology is only but a driver of available content and hence it thrives, and finds usefulness in the context of information, particularly transformative evidence for universal global health resources access and sharing benefits for all.\n\nHealth financing or national resource allocation requires as much open data and information access, analysis, effective and reliable interpretation for outcomes-based sustainable and equitable early decision health financing and funding in achieving local and regional Universal health coverage (UCH) and sustainable development goals (SDGs). Moreover, this new paradigm has the capacity to strengthen and allow exploration of potential local and national health systems, insurance schemes implementation, uptake and coverage impacts as well as legislative and institutional reforms and regulations to enable community and stakeholders commitments and investments13,19.\n\n\nImplications of strengthening knowledge-based innovations and health systems in Africa\n\nProactive efforts in promoting radical data and information openness and defining criteria tailored to sharing capabilities and transparency are critical and innovative approaches to create monetary and non-monetary benefits13,16. Data-driven approaches and strategies provide an immense opportunity to understand, define and generate databases that can be used for predictive primary care and innovations in the short- and long-term in diverse scenarios14,16. The substantiation is argued by knowledge-based open data or information access platforms and proven models that are urgently needed in promoting useful and efficient public health intelligence, health programming and ample financial allocation in disease prevention and control in line with the Abuja declaration in 2000 and health for all of the Africa Union vision 2063 (AU, 20163) and Pan-Africanism agenda13,14,17,20,21.\n\nIn order to attain and optimize the Pan-Africanism aspiration to FOA to current findings and evidence that help shape our decision making process, it is imperative to consolidate online platforms and resources to one stop shop for evidence in different genres. Since timely access to accurate data and information are essential to improving the quality of knowledge and intervention effectiveness, to information scientists alongside librarians globally. Predominantly, with the challenges of electricity shortage and costly internet services, most open access African users’ tendency is increasingly familiar with Google and other internet search engines to discover or access information. Hence, any FOA platform should be user-friendly, non-bias choice, interoperability and flexible. That is, accessibility should not depend on articles being accessed via a special portal or proxy server or publishing platforms, or via complex authorization systems, but should be readily and freely available to all re-users or users consumers and redistribution within the ethical and legal framework. Information should be readily reached without barriers targeting all cadres of technocrats from those with basic training and skills to the advanced. Tested models have proved this relevance and lesson for progressive improvement can be adopted16,17,22. One of the implications of not doing so is not being able to find information easily using online systems14,22,23. Consequently, researchers and policy-makers and implementers in LMICs have to spend enough time to be informed, consolidate and synthesize what types of information and knowledge can be adaptable, scalable, cost-effective and translated in intervention and best practices14,22,23. As a way out in many occasions, when we develop policies for research and programs delivery, we as institutions or individuals take slightly different routes to find the evidence that helps shape our decision making. We often end up relying on a restricted range of platforms, consortia systems and institutional networks that are only readily available small scale data and database evidence online20. Hence, without objectivity and in absence of credible context relevant platforms, we are prone to use biased and/or misplaced approaches policies particularly in the health systems improvement endeavor14,20,23.\n\n\nHarnessing FOA and resources sharing advocacy and mitigation platforms and policies\n\nFOA platforms prospects are multiple to African scholars, researchers and their collaborators real time and frontline data and other research outputs contextual determinants and scenarios will preferentially entail consolidating R&Ds that are alternatives to the prevailing publishing proprietary models to support open access to health resources. For instance, prioritization databases combine available genomic, genetics and bioinformatics data for each priority genre with automatically extracted and manually curated information for genetic counseling to personalized medicine. Also, in questioning or responding to further literature and other databases research gaps relevant to clinical and analytical practices, putative drug and vaccine target(s) discovery for threatening chronic diseases. Investing substantial efforts in open database mining also permits prioritization, actionable and customized evidence, potential drug and vaccine targets discovery3,4,11,18. Such harnessing may entail the development of research and innovation portfolios focusing on critical public health gaps where traditional approaches are failing, and leveraging proven evidence and lessons learnt on what works and what does not work. As such, the aim would be to attain a long term health agenda and capacity building mitigation via research approaches, cost-effective, timely and progressive innovations24,25.\n\nAuthors advocate to governments, policy-makers and implementers, researchers, academicians, health professionals and other stakeholders including the community to endorse open access public health resources platforms implementation at all levels. There is also need to develop appropriate mechanisms and strategies to promote open access capacity building and empowerment, enhanced health and disease literacy and education through sharing and exchanges, innovative policies and frameworks with advances in digital technologies, establishment of data and information quality control and assurance principle and guidelines, well-coordinated and coherent metadata and database management for evidence operational research and clinical decision making interventions26,27. The value and credit of FOA does not only promote health and disease literacy, but offer opportunities for mutual sharing of various educational materials, learning and empowerment on maximizing on the use or reuse of the data mining and managed for short- and long-term public health benefits, global health security and wellbeing.\n\nFOA agenda to health workers, professional and providers and communities offers new opportunities in providing affordable, robust, real time and free user friendly and sustainable datasets and databases13,18,24,25. While proactive efforts in reducing or minimizing the various barriers and challenges of FOA uptake and implementation capacity including intellectual property rights, confidentiality, legislation and data use agreements amongst stakeholders (including the lay communities, institutions of learning and studentships) still persist, the value of open access is real13,16,18,24.\n\n\nOpen access resources and public health benefits in sub-Saharan Africa\n\nThe value of freely accessible and available scarce and/or other profuse data, database and information through FOA for public health systems offers tremendous opportunities to strengthen and fasten emerging threat and epidemics including persistent infectious diseases of poverty modeling in preparedness, prevention and control. Moreover, promoting robust evidence-based health and disease surveillance, response planning and funding underscore the social, ecological and economic burden, and opportunities for governments, stakeholders and vulnerable populations13,19,20,22,26,27. Nonetheless, FOA and information sharing potential benefits and gains should include but not limited to:\n\n(1) Enhancing new public health paradigm and innovations in collective and participative education, timely reporting and increasing dissemination and effective trans-boundary risk communication towards democratization of heath data and information for quality health and wellbeing.\n\n(2) Accelerating proven acceptability and uptake tactics and sustainable mechanisms such as expanded vaccine(s) immunization or mass drug administration in scaling up the coverage and effectiveness to prevent disability and death; uses of wearable technology and sensors in early detection, tracking and monitoring of vectors and/or pathogens and management of associated diseases including non-communicable diseases mitigation and lifestyle adaptations strategies.\n\n(3) Upholding continuous open access resources advocacy, education and awareness for all in securing universal health coverage, SDGs and “FOA health information for all generations”.\n\n(4) Nurturing new commitment and investment in novel proven approaches, methods and tools in strengthening local and regional health systems capacity development (infrastructures and resources) in operational and translation research from diverse resources and sources.\n\n(5) Promoting the value of free, real-time data and information access and availability to all parties in transforming knowledge-based translation into health policy decisions and guiding health priorities financing and public health actions.\n\n(6) Improving integration and use of information to support evidence-based integrated public-private health and related sectors partnerships (local private sector, bilateral and multi-international) and community-based programs and projects participative ownership.\n\n(7) Fostering innovative interventions and best practices amongst professional, health workers and the community resilience and participative engagement in response to emerging threats and disasters.\n\n(8) Facilitating lifetime interactive learning, increasing knowledge, empowerment and resilience in emerging epidemics and infectious diseases vulnerability surveillance and monitoring measures.\n\n(9) Promoting ethical, legal and international regulations and by-laws applications in safety and security.\n\n(10) Promoting local, national and regional “One Health” approach in tackling in integrated manner regional and global epidemics of zoonotic infectious diseases prevention, preparedness, control and elimination agenda integration, uptake and utilization for impact.",
"appendix": "Author contributions\n\n\n\nET conceived the idea, researched the literature and initiated the primary draft of the manuscript. ET, GM and CKW contributed to review the literature. ET, GM, CKW, ENT, OAO, NM, LB, AAA and JYN provided further inputs. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nChattopadhyay J: Are Press Depictions of Affordable Care Act Beneficiaries Favorable to Policy Durability? Politics Life Sci. 2015; 34(2): 7–43. PubMed Abstract\n\nSolomon GM, Morello-Frosch R, Zeise L, et al.: Cumulative Environmental Impacts: Science and Policy to Protect Communities. Annu Rev Public Health. 2016; 37: 83–96. PubMed Abstract | Publisher Full Text\n\nNick J: Open access part II: The structure, resources, and Implications for nurses. Online J Issues Nurs. 2011; 17(1): 7. PubMed Abstract\n\nBecnel LB, Pereira S, Drummond JA, et al.: An open access pilot freely sharing cancer genomic data from participants in Texas. Sci Data. 2016; 3: 160010. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeone T, Cetorelli V, Neal S, et al.: Financial accessibility and user fee reforms for maternal healthcare in five sub-Saharan countries: a quasi-experimental analysis. BMJ Open. 2016; 6(1): e009692. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoneyman A, Cox B, Fisher B: Potential impacts of patient access to their electronic care records. Inform Prim Care. 2005; 13(1): 55–60. PubMed Abstract | Publisher Full Text\n\nGenerous N, Fairchild G, Deshpande A, et al.: Global disease monitoring and forecasting with Wikipedia. PLoS Comput Biol. 2014; 10(11): e1003892. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOniscu GC, Ravanan R, Wu D, et al.: Access to Transplantation and Transplant Outcome Measures (ATTOM): study protocol of a UK wide, in-depth, prospective cohort analysis. BMJ open. 2016; 6(2): e010377. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDzakpasu S, Soremekun S, Manu A, et al.: Impact of free delivery care on health facility delivery and insurance coverage in Ghana’s Brong Ahafo Region. PLoS One. 2012; 7(11): e49430. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcIntyre KM, Setzkorn C, Wardeh M, et al.: Using open-access taxonomic and spatial information to create a comprehensive database for the study of mammalian and avian livestock and pet infections. Prev Vet Med. 2014; 116(3): 325–335. PubMed Abstract | Publisher Full Text\n\nKostkova P, Fowler D, Wiseman S, et al.: Major infection events over 5 years: how is media coverage influencing online information needs of health care professionals and the public? J Med Internet Res. 2013; 15(7): e107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRogers A, Vassilev I, Brooks H, et al.: Brief encounters: what do primary care professionals contribute to peoples’ self-care support network for long-term conditions? A mixed methods study. BMC Fam Pract. 2016; 17(1): 21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMills A: Strengthening health systems: the role and promise of policy and systems research. World health organization (WHO). Alliance for health policy and systems research; 2004. Reference Source\n\nCockerill MJ, Knols BG: Open access to research for the developing world. Issues Sci Technol. 2008; 24(2): 65. Reference Source\n\nAwoonor-Williams JK, Sory EK, Nyonator FK, et al.: Lessons learned from scaling up a community-based health program in the Upper East Region of northern Ghana. Glob Health Sci Pract. 2013; 1(1): 117–133. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeonard KL: Active patients in rural African health care: implications for research and policy. Health Policy Plan. 2014; 29(1): 85–95. PubMed Abstract | Publisher Full Text\n\nAronson B: Improving online access to medical information for low-income countries. N Engl J Med. 2004; 350(10): 966–968. PubMed Abstract | Publisher Full Text\n\nFrenk J, Chen L, Bhutta ZA, et al.: Health professionals for a new century: transforming education to strengthen health systems in an interdependent world. Lancet. 2010; 376(9756): 1923–1958. PubMed Abstract | Publisher Full Text\n\nOrganization WH: Strengthening health systems to improve health outcomes. WHO's Framework for Action, 2007. In.; 2013. Reference Source\n\nPowell M: Which knowledge? Whose reality? An overview of knowledge used in the development sector. Dev Pract. 2006; 16(6): 518–532. Publisher Full Text\n\nJain P: Promoting open access to research in academic libraries. 2012. Reference Source\n\nCzerniewicz L, Goodier S: Open access in South Africa: A case study and reflections. S Afr J Sci. 2014; 110(9–10): 01–09. Publisher Full Text\n\nNaude CE, Zani B, Ongolo-Zogo P, et al.: Research evidence and policy: qualitative study in selected provinces in South Africa and Cameroon. Implement Sci. 2015; 10(1): 126. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSwanson RC, Atun R, Best A, et al.: Strengthening health systems in low-income countries by enhancing organizational capacities and improving institutions. Global Health. 2015; 11(1): 5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChu KM, Jayaraman S, Kyamanywa P, et al.: Building research capacity in Africa: equity and global health collaborations. PLoS Med. 2014; 11(3): e1001612. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTickell A: Open access to research publications: independent advice. 2016. Reference Source\n\nKerridge S, Ward P: Open access for REF2020. Insights. 2014; 27(1): 58–62. Publisher Full Text"
}
|
[
{
"id": "14072",
"date": "31 May 2016",
"name": "Oyewale Tomori",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 addressed the highly important issue of free open access (FOA) as a means of tackling emerging epidemics and infectious diseases burden in Africa. They argue that unrestricted open access and free use or reuse of relevant and appropriate data together with sharing of data among African researchers, international partners, policy makers and the community will help limit the effect of burden of infectious disease epidemics in Africa. Availability of real time information on virtually all aspects of infections and diseases is required for timely action and response needed to ensure that public health events do not escalate to international concerns with grave consequences. It must be however observed that unrestricted free open access to data may lead to users being overwhelmed and unable to make sense out of the data, unless there is available capacity and capability for appropriately analysing and synthesizing the data into valuable information for policy-makers, implementers, care providers, researchers, health-related institutions and stakeholders including the community. This requires increasing and improving human resource capacity side by side with the call for unrestricted free open access. I find the review stimulating and challenging, with title and abstract matching the contents and conclusions which focuses on the benefits of FOA. As they call for FOA, they also call for the need for unrestricted but planned collaboration between all stakeholders under the “One Health” umbrella.",
"responses": []
},
{
"id": "14730",
"date": "15 Jul 2016",
"name": "Daniel Achinko",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 was carefully thought of by the authors in relation to the current disease burden faced by the world and its people. The concept of FOA as a driving force for better networking on the knowledge of disease burden, has been an area of concern by researchers, health organizations and the various government bodies around the world. Research based information generated by different labs using high throughput information has been the driving force for obtaining large funding, reasons why the published information cannot be freely accessed in some journals. The resulting challenge is the lack of implementation of the right approaches to disease outcomes in LMICs due to low comprehension of the disease epidemiology and genetic factors. There is actually need for world governing bodies to sit at their conferences and make FOA a priority so that researchers in LMICs, through documented information in accessed journals, could collaborate with authors who have published widely in related fields of interest, helping them contain the disease epidemic using the right approaches before it spreads. The world now is a global village and should be treated as such, hence institutions at all ends of the globe should be able to create some form of partnerships for training and sharing data for the advancement of better health for all.\nThe authors actually focused on their indicated topic and brought out related challenges, which suggests reasons for poor approaches to the current disease burden. Their proposals highlight steps which every player involved in decision making at various levels of the disease burden for better health, need to consider for us to attain the health goals put in place.\nI recommend this article as a document for all involved in the research and health field.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-853
|
https://f1000research.com/articles/5-852/v1
|
09 May 16
|
{
"type": "Review",
"title": "Healing through language: Positive physical health effects of indigenous language use",
"authors": [
"D. H. Whalen",
"Margaret Moss",
"Daryl Baldwin",
"Margaret Moss",
"Daryl Baldwin"
],
"abstract": "This article summarizes existing work that indicates language maintenance and revitalization efforts result in health-related benefits for Native Americans and other indigenous populations. Although forced loss of ancestral language has been a feature of life in most indigenous communities since the first contact with Europeans, the pace of loss has accelerated in the past 50 years. Among the many hardships such communities face, an especially troubling one is lowered health status. There are indications, however, that language maintenance and revitalization efforts have positive effects on physical and communal health among indigenous populations. The types of language programs currently in place are outlined along with a variety of studies that will measure health improvement outcomes correlated with language revitalization efforts. Such evidence justifies increased support for language revitalization in order to improve health.",
"keywords": [
"indigenous health",
"social determinants of health",
"language",
"Native American",
"resilience",
"revitalization",
"community"
],
"content": "Introduction\n\nIndigenous communities have some of the worst health outcomes within their larger societies (Gracey & King, 2009). This is true across the world, for example, in Australia and New Zealand (Anderson et al., 2006), Latin America (Pan American Health Organization, 2001), Canada (Frohlich et al., 2006), and the United States (U.S. Commission on Civil Rights, 2004). Although many factors, such as location, poverty, and access to health facilities, contribute to this disparity, there is a growing recognition that historical trauma plays a role as well (Duran et al., 1998; Sotero, 2006). The present survey examines published studies that address one salient means of recovering from historical trauma, namely, maintenance and revitalization of indigenous language. These studies consistently find that there are physical health advantages to be gained from such efforts.\n\nThe ways in which languages emerge, evolve, and fall dormant have been increasingly discussed by linguists, biologists and cognitive scientists in the last two decades (Hale et al., 1992; Wang & Minett, 2005). However, health scientists have not largely embraced this discussion as relevant. Although native language is sometimes mentioned in \"health literacy\" (e.g. Berkman et al., 2011), health related outcomes as a result of language maintenance and revitalization efforts have not received much attention. Here, we explore the limited existing literature on health outcomes of indigenous language use and outline promising research avenues for the future.\n\nLanguage is a uniquely efficient means of maintaining and passing on crucial cultural knowledge and thus promotes cultural diversity (McCarty & Zepeda, 2010). Although languages have come and gone throughout history, indigenous languages in a variety of post-colonial situations have been oppressed through official policies and social pressures (Dussias, 1999; Oster et al., 2014). These forced language shifts caused the loss of social and behavioral knowledge and understanding that was created over millennia, ultimately contributing to disastrous, endemic health deficits (Gracey & King, 2009; Hallett et al., 2007; King et al., 2009).\n\nEmerging evidence (see “Existing studies” section below) ties individual and/or communal health to efforts that promote ancestral language and cultural fluency in a community-driven educational environment. Some of the literature addresses indigenous groups affected by the four settler states of the United States (US), Canada, New Zealand and Australia. The similarities in colonialism, history and outcomes for the original peoples outweigh the inevitable differences in specific situations. Further, the political boundaries between Canada and the US do not reflect original use and are often unrecognized by the first peoples. Here, indigenous people of North America (hereafter, called Native Americans or (NA)) will be an exemplar as we explore the ability to use one’s ancestral language as a knowledge-seeking catalyst to optimize health.\n\n\nHealth status\n\nNative Americans suffer some of the poorest health statuses and health outcomes of any other racial group in the US (Indian Health Service, 2014). This includes shorter lifespans, higher death rates from a wide variety of causes, and more limited access to health services. According to the 2010 American Community Survey, NA have a higher rate of disability than any other single group at 15%, almost 25% higher than for whites. While some of this is due to rural poverty, disparities are profound even within urban areas (as reported for Canada) (Firestone et al., 2014).\n\nCurrent, low socio-economic status clearly is a major factor for poor health outcomes in NAs, but NA health today also results from historical trauma and its inter-generational continuation. Federal Indian Policies from 200 years of removal, reservations, relocation/assimilation, termination, and language eradication have resulted in loss of identity, shame, guilt, unresolved grief and depression (Brave Heart & DeBruyn, 1998; Oster et al., 2014), key features of historical trauma. In today’s Native America, extreme poverty, poor health, and high homicide and suicide rates are tragic outcomes of this historical trauma (Brave Heart & DeBruyn, 1998; Sotero, 2006).\n\n\nIndigenous language loss and language use\n\nThe loss of languages worldwide has been substantial over the past 200 years. Many linguists estimate that half to 90% of the world’s languages will be moribund by the end of this century (Grenoble & Whaley, 1998; Hale et al., 1992; Whalen & Simons, 2012). Even so, there was little concentrated effort to retain and revitalize language among NA communities until recently, aided by the passage of the Native American Languages Act of 1990. Despite subsequent progress, NA languages are still rapidly losing speakers and the knowledge residing in their languages.\n\nLanguage and culture are intertwined, and neither completely determines the other (McCarty & Zepeda, 2010), but language use is a substantial indicator of cultural coherence. Many NA communities are engaged in the revival of language and culture (see examples Table 1), and raising the health of the community is often an explicit goal. For example, Stephanie Fielding (Mohegan Tribe, Council of Elders), states, “The Mohegan language program is more than a cultural effort, it is aimed at bringing our community together to improve its cohesion and health” (personal communication, 20 April 2016). There are indications that such efforts can be successful (e.g., Hinton, 2001:225), but the next section will survey published studies that more directly assess the effects of language maintenance (i.e., for first language (L1) speakers) and language revitalization (i.e., for second language (L2) learners).\n\nL1/L2 indicates whether the language is a first language (L1) or learned later in life (L2) for the target population. Ratings of “poor” are often due to small numbers available.\n\n\nExisting studies\n\nNative health, from their own perspective, includes four domains: spiritual, mental, emotional and physical; a disruption in any part, is a loss in ‘health’ (Moss, 2015). More often there are several domains affected, so each must be considered in this discussion.\n\nSpiritual life has always been key for American Indians. Intergenerational trauma or historical trauma has been termed as a ‘spiritual injury’, or ‘soul wound’. A large driver of this trauma was the taking away of language (Duran et al., 1998). Prayers and ‘medicine men’ have utilized indigenous languages traditionally (Moss, 2000). In many cases it is the spirit that is being healed.\n\nWhile the present survey focuses on the physical effects, mental and emotional factors certainly play a role in suicide. Hallett et al. (2007) found the youth suicide rate for indigenous bands in British Columbia in which 50% of the community is conversationally fluent (L1) to be 1/6th that of less fluent bands. The language factor was predictive even when other correlated cultural factors were subtracted from the analysis.\n\nOn the physical side, another study found that the percentage of smokers in US Southwestern tribes, where indigenous languages are widely spoken, was only 14 percent as compared to 50 percent among Northern Plains tribes, where language use is sparse (Nez Henderson et al., 2005). A survey study in California (Hodge & Nandy, 2011:797) found that those who could speak their ancestral language were more frequently classified in a (within-study defined) “good” wellness group (82.4%) than were those who could not speak it (70.1%). A survey of residents of the Hopi reservation found that language use was positively correlated with “practicing Hopi behaviors to keep healthy” (Coe et al., 2004:405). A recent study found that Canadian First Nations groups with greater cultural retention, as indexed by language use, had significantly lower rates of diabetes after factoring out socioeconomic factors (Oster et al., 2014). All of these studies combined L1 and L2 speakers.\n\nIn Australia, speaking an Aboriginal and/or Torres Strait Islander language was found to reduce several health risk factors such as excessive alcohol consumption (8% vs. 18% for English monolinguals), illicit drug use (16% vs. 26%), and violence victimization (25% vs. 37%) (Australian Institute of Aboriginal and Torres Strait Islander Studies (AIATSIS), 2005:3). Those who spoke their heritage language were also found to have half the rate of “poor health” as those who had switched to English (Trewin & Madden, 2005:95). Alcohol, violence and drug use (especially methamphetamines) are problematic in NA populations at rates exceeding the general US population (IHS, 2014:191).\n\nOne result based exclusively on L2 learners examined high school graduation rates, which are taken as a health indicator by the National Institutes of Health, in a Hawaiian language immersion school. The Nāwahī program in Hawaii has a 100% graduation rate (Wilson, 2012), compared to 70% for standard schools (Kamehameha Schools, 2009).\n\n\nWhy focus on language?\n\nLanguage transmission is a particularly effective means of reinforcing culture and has the benefit of integrating most cultural and communal activities. Language is also an extremely efficient means of establishing membership or inclusion in a community. Studies that might disentangle language and culture are possible, but the approach argued for here takes it as a given that language is the most efficient means of transmitting, maintaining, and even reviving culture.\n\nThere are different kinds of environments and programs for language learning and use (Table 1). Evaluation can be a challenge, whether the study is retrospective or prospective, due to large differences in specifics and small numbers in many cases. Census data can, with limitations, add to the results. Prospective studies could compare such health measures as absentee rates from school or changes in the Youth Risk Behavior Surveillance System (YRBSS; Centers for Disease Control and Prevention, 2004). While indirect, these measures can be obtained for all children, allowing larger cohorts than would be possible if only eventual negative outcomes were measured.\n\nThe main recommendation suggested by these studies, then, is that language programs prospectively include assessment of health outcomes along with the core of the program (see also Marmion et al., 2014:46). In this way, samples across the wide variety of programs can be compared, with the expectation that those with the most positive health outcomes can be replicated in other programs. Comparisons, both in effectiveness and cost, to standard health programs are also needed, given that language programs are typically less expensive than many other types of programs.\n\n\nConclusion\n\nLanguage programs in NA communities hold the promise of improving the mental and physical health of those who participate in them. All generations seem able to take advantage of this possibility. Data exist on palliative effects of use of a Native language and/or cultural practices on smoking (Nez Henderson et al., 2005), suicide rates (Hallett et al., 2007) and alcohol (Torres Stone et al., 2006; Whitbeck et al., 2004) and substance abuse (First Peoples’ Heritage Language and Culture Council, 2012:213), but many more issues are likely to be evident as more data are collected. A return to native spirituality has been shown to improve treatment results (Berry et al., 2012; Gone & Calf Looking, 2015), an effect that could be expected to increase with inclusion of the ancestral language. As Tulloch et al. (2013) point out, the many informal programs that have appeared recently are not well studied and may require novel methodologies.\n\nThe Endangered Language Fund has launched a program called Healing Through Language to facilitate the support and assessment of language programs throughout Native America (www.healingthroughlanguage.org). Communities throughout the US and elsewhere have started language programs with a wide range of models. Because this one type of program can, apparently, lead to positive outcomes in so many areas, we would expect to find a substantial return in health benefits on investment in language programs in Native America.\n\nSpeaking an indigenous language in and of itself may not be solely responsible for improved health status; rather, community validation of the indigenous knowledge system, community-driven tribal education, comprehending indigenous medicine, and youth empowerment through language and culture identity formation all play significant roles in the ability of a minority language to thrive and for the community to experience healthy outcomes from the collective effort.",
"appendix": "Author contributions\n\n\n\nAll authors reviewed the literature and provided input to and reviewed all drafts of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nHelpful comments were provided by Monica Macaulay, Alice Taff, Susan Penfield and Linda Mayes.\n\n\nReferences\n\nAnderson I, Crengle S, Kamaka ML, et al.: Indigenous health in Australia, New Zealand, and the Pacific. Lancet. 2006; 367(9524): 1775–1785. PubMed Abstract | Publisher Full Text\n\nAustralian Institute of Aboriginal and Torres Strait Islander Studies (AIATSIS): National Indigenous Languages Survey Report 2005. 2005. Reference Source\n\nBerkman ND, Sheridan SL, Donahue KE, et al.: Low health literacy and health outcomes: an updated systematic review. Ann Intern Med. 2011; 155(2): 97–107. PubMed Abstract | Publisher Full Text\n\nBerry SL, Crowe TP, Deane FP, et al.: Growth and empowerment for Indigenous Australians in substance abuse treatment. Int J Ment Health Addict. 2012; 10(6): 970–983. Publisher Full Text\n\nBrave Heart MY, DeBruyn LM: The American Indian Holocaust: healing historical unresolved grief. Am Indian Alsk Native Ment Health Res. 1998; 8(2): 60–82. PubMed Abstract | Publisher Full Text\n\nCantoni G(Ed.): Stabilizing indigenous languages. Flagstaff, AZ: Northern Arizona University Press. 1996. Reference Source\n\nCenters for Disease Control and Prevention: Methodology of the youth risk behavior surveillance system. MMWR Surveill Summ. 2004; 54(RR-12): 1–13. Reference Source\n\nCoe K, Attakai A, Papenfuss M, et al.: Traditionalism and its relationship to disease risk and protective behaviors of women living on the Hopi reservation. Health Care Women Int. 2004; 25(5): 391–410. PubMed Abstract | Publisher Full Text\n\nDuran B, Duran E, Brave Heart MYH: The trauma of history. In R. Thornton (Ed.), Studying Native America: Problems and perspectives. Madison, WI: University of Wisconsin Press. 1998; 60–78. Reference Source\n\nDussias AM: Waging war with words: Native Americans' continuing struggle against the suppression of their languages. Ohio State Law Journal. 1999; 60(3): 901–993. Reference Source\n\nFirestone M, Smylie J, Maracle S, et al.: Unmasking health determinants and health outcomes for urban First Nations using respondent-driven sampling. BMJ Open. 2014; 4(7): e004978. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFirst Peoples’ Heritage Language and Culture Council: Report on the Status of B.C. First Nations Languages 2010. Brentwood Bay, B.C.: First Peoples’ Heritage, Language and Culture Council. 2012. Reference Source\n\nFrohlich KL, Ross N, Richmond C: Health disparities in Canada today: some evidence and a theoretical framework. Health Policy. 2006; 79(2–3): 132–143. PubMed Abstract | Publisher Full Text\n\nGone JP, Calf Looking PE: The Blackfeet Indian culture camp: Auditioning an alternative indigenous treatment for substance use disorders. Psychol Serv. 2015; 12(2): 83–91. PubMed Abstract | Publisher Full Text\n\nGracey M, King M: Indigenous health part 1: determinants and disease patterns. Lancet. 2009; 374(9683): 65–75. PubMed Abstract | Publisher Full Text\n\nGrenoble LA, Whaley LJ (Eds.): Endangered languages: Language loss and community response. Cambridge: Cambridge University Press. 1998. Reference Source\n\nHale K, Krauss M, Watahomigie LJ, et al.: Endangered languages. Language. 1992; 68(1): 1–42. Publisher Full Text\n\nHallett D, Chandler MJ, Lalonde CE: Aboriginal language knowledge and youth suicide. Cognitive Development. 2007; 22(3): 392–399. Publisher Full Text\n\nHinton L: The master-apprentice language learning program. In L. Hinton & K. Hale (Eds.), The Green Book of language revitalization in practice. London: Academic. 2001; 217–226. Publisher Full Text\n\nHodge FS, Nandy K: Predictors of wellness and American Indians. J Health Care Poor Underserved. 2011; 22(3): 791–803. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIndian Health Service: Trends in Indian health: 2014 edition. Rockville, MD: Indian Health Service. 2014. Reference Source\n\nKamehameha Schools: Native Hawaiian educational assessment update 2009: A supplement to Ka Huaka‘i 2005. Honolulu: Kamehameha Schools, Research and Evaluation Division. 2009. Reference Source\n\nKing M, Smith A, Gracey M: Indigenous health part 2: the underlying causes of the health gap. Lancet. 2009; 374(9683): 76–85. PubMed Abstract | Publisher Full Text\n\nLes Whitbeck B, Chen X, Hoyt DR, et al.: Discrimination, historical loss and enculturation: culturally specific risk and resiliency factors for alcohol abuse among American Indians. J Stud Alcohol. 2004; 65(4): 409–418. PubMed Abstract | Publisher Full Text\n\nMarmion D, Obata K, Troy J: Community, identity, wellbeing: The report of the Second National Indigenous Languages Survey. Canberra: Australian Institute of Aboriginal and Torres Strait Islander Studies (AIATSIS). 2014. Reference Source\n\nMcCarty TL, Zepeda O: Native Americans. In J. A. Fishman & O. García (Eds.), Handbook of language and ethnic identity. (2nd ed) Oxford; New York: Oxford University Press. 2010; 323–339.\n\nMoss MP: Zuni elders: Ethnography of American Indian aging. (Ph.D. dissertation), University of Texas School of Nursing at Houston. 2000. Reference Source\n\nMoss MP: American Indian health and nursing. New York: Springer. 2015. Reference Source\n\nNez Henderson P, Jacobsen C, Beals J, et al.: Correlates of cigarette smoking among selected Southwest and Northern plains tribal groups: the AI-SUPERPFP Study. Am J Public Health. 2005; 95(5): 867–872. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOster RT, Grier A, Lightning R, et al.: Cultural continuity, traditional Indigenous language, and diabetes in Alberta First Nations: a mixed methods study. Int J Equity Health. 2014; 13(92): 1–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPan American Health Organization: Equity and health: Views from the Pan American Sanitary Bureau. Washington, DC: PAHO. 2001. Reference Source\n\nSotero MM: A conceptual model of historical trauma: Implications for public health practice and research. J Health Dispar Res Pract. 2006; 1(1): 93–108. Reference Source\n\nStone RA, Whitbeck LB, Chen X, et al.: Traditional practices, traditional spirituality, and alcohol cessation among American Indians. J Stud Alcohol. 2006; 67(2): 236–244. PubMed Abstract | Publisher Full Text\n\nTrewin D, Madden R: The health and welfare of Australia’s Aboriginal and Torres Strait Islander peoples. Canberra: Australian Bureau of Statistics. 2005. Reference Source\n\nTulloch S, Kusugak A, Uluqsi G, et al.: Stitching together literacy, culture & well-being: The potential of non-formal learning programs. Northern Public Affairs. 2013; 2(2): 28–32. Reference Source\n\nU.S. Commission on Civil Rights: Broken promises: Evaluating the Native American health care system. Washington, DC: U.S. Commission on Civil Rights. 2004; 1–155. Reference Source\n\nWang WS, Minett JW: The invasion of language: emergence, change and death. Trends Ecol Evol. 2005; 20(5): 263–269. PubMed Abstract | Publisher Full Text\n\nWhalen DH, Simons GF: Endangered language families. Language. 2012; 88(1): 155–173. Publisher Full Text\n\nWilson WPH: USDE violations of NALA and the testing boycott at Nāwahīokalani'ōpu'u School. J Am Indian Educ. 2012; 51(3): 30–45. Reference Source"
}
|
[
{
"id": "14162",
"date": "07 Jun 2016",
"name": "Richard T Oster",
"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 timely and important article. Indigenous peoples continue to voice the importance of culture and language revitalization for their well-being. As a result, more and more research is being conducted linking language and health in Indigenous populations. A review or summary of that work is needed, hence the current article. I commend the authors for undertaking this vital task. Generally, the article is well written and informative. However, I found that some sections were not thoroughly explored and the authors would do well by deepening their literature review and expanding on their ideas. My comments are as follows:\nI suggest the authors capitalize the term “Indigenous” throughout the article.\n\nAbstract, third sentence: it is not clear what the authors mean by “communities” here. Also, compared to what population is their “lowered health status”?\n\nAbstract: it would be useful to add a sentence on how language revitalization is thought to improve health.\n\nIntroduction: the authors state that this current article is a survey. This is a bit confusing. The authors should be clear on what type of article this is. It is not a quantitative survey, systematic review, meta-analysis, review, or literature search. As the article is now, an “opinion piece” seems more appropriate. However, I’m hoping that the authors can expand on many of their ideas and explore the known literature further. In which case, the article may be appropriately termed a “review”.\n\nIntroduction, paragraph 3, last sentence: I’m not sure that the references cited support the claim being made that language shifts have caused the health deficits we see today among Indigenous populations. Perhaps the authors can rework this sentence.\n\nHealth status: this is one of the sections that seems lacking in its scope. Perhaps the authors can expand and describe some of the major health disparities among Indigenous populations. Some epidemiological data would be helpful to the reader to understand the scope of the disparities.\n\nIndigenous language loss and language use: it would be helpful to the reader if the authors provided examples of some of the Indigenous languages. What are some of the major Indigenous languages worldwide? How many are there? How many have been lost? It would also be helpful if the authors provided some data on how much Indigenous languages are being spoken today around the world and in North America.\n\nExisting studies and why focus on language sections: These sections in particular need to be expanded upon. Are the authors able to conduct a formal literature search? Perhaps with keywords? Also, the authors may want to explore and include other literature that has explored the relationship between language/culture and emotional/mental health. The extensive writings of Laurence Kirmayer come to mind as a good place to start. What is explicitly missing from this article is the how piece... How does language revitalization lead to better health? Is it due to enhanced sense of identity (as Chandler and Lalonde argue)? Enhanced spiritual health? Enhanced sense of community/belonging? The provision of strong role models that teach the language? Protection from early life traumas? There is also an entire field of neurological research that has looked at how learning multiple languages can impact the brain and health. The authors would do well to explore these different areas and give the reader an idea on the how. Even looking into some of the qualitative research with Indigenous peoples suffering from some of the major health problems that Indigenous populations face (e.g. diabetes) would be beneficial as often language/culture is brought up as a key determinant of health in these studies. Moreover, has there been research that has shown not shown a relationship between language and health? Lastly, second paragraph, last sentence: Has there been work done that specifically examined language loss and sense of spiritual well-being?\n\nI was pleased that the authors recommend that future studies aimed at language revitalization include assessments of health outcomes. Perhaps the authors could expand on what these health assessments may look like? What should be measured and how?\n\nConclusion: It would make more sense perhaps to include and expand upon the Berry 2012, Gone 2015, and Tullouch 2013 references in the “existing studies” or “why focus on language” sections.\n\nLast paragraph of conclusion: the ideas presented in this paragraph seem to come out of nowhere as they have not been discussed earlier in the article. Again, the authors would do well to really spend some time reading and then summarizing in the “existing studies” or “why focus on language” sections the known literature on how and why language revitalization may lead to improved health. These sections are the place (as opposed to the conclusion) to introduce and describe in-depth the other contributors related to speaking an Indigenous language that may be responsible for improved health status.",
"responses": []
},
{
"id": "14157",
"date": "21 Sep 2016",
"name": "Terryann Coralie 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\nThank you for the opportunity to review this review.\nI think this article is of significant importance for indigenous peoples and links ancestral wisdom with some evidence. I do think that the article is weak in some areas:\nAbstract I think the abstract could stronger. It is unclear what you conclude and what evidence you have. e.g. Evidence suggests that language maintenance can be associated with improved health, social and education outcomes for American Indians an indigenous populations. This is evidenced by ...\nIntroduction\n\nThe links between language and well-being need to be stronger. Need more evidence that language is associated with well-being. What is the theories about how language and cultural maintenance, cultural connection improve outcomes? Think your paper would be stronger if it maintained its international literature focus - there is more evidence if you use Australia, NZ and other indigenous people. Need to state what the purpose of the paper is. What you intend to review.\nHealth status This section could be stronger. There is a lot of evidence about poor health among indigenous peoples. What is health from an indigenous perspective?\nIndigenous language and loss Talking about 'language loss' suggests that it was somehow accidental. Language was a purposeful way to colonise indigenous peoples.\n\nExisting studies (Evidence to support language revitalisation?) Not sure about this heading - what is meant by existing studies? Watch causation language - these are associations e.g. \"speaking an indigenous language was found to reduce several health risks (p.3) \" - should read speaking and indigenous language was associated with reduced risk.\nWhy focus on language? Think this section should come earlier - what is the theory that language is a powerful protective factor? The table provided is unrelated to the content and doesn't add much. Unsure what this is for - link to purpose statement at the beginning. e.g. The purpose of this review is to review the literature about the importance of language to indigenous well-being and identify the programmes and data available that might be evaluated? Need more on this as it is not clear from your review.\nConclusions Should be more focused.\n\nOverall - I think this is useful information for indigenous peoples but it requires a significant restructure and rewrite to make it cohesive.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-852
|
https://f1000research.com/articles/5-825/v1
|
09 May 16
|
{
"type": "Research Note",
"title": "Assessment of pharmacogenomic agreement",
"authors": [
"Zhaleh Safikhani",
"Nehme El-Hachem",
"Rene Quevedo",
"Petr Smirnov",
"Anna Goldenberg",
"Nicolai Juul Birkbak",
"Christopher Mason",
"Christos Hatzis",
"Leming Shi",
"Hugo JWL Aerts",
"John Quackenbush",
"Benjamin Haibe-Kains",
"Zhaleh Safikhani",
"Nehme El-Hachem",
"Rene Quevedo",
"Petr Smirnov",
"Anna Goldenberg",
"Nicolai Juul Birkbak",
"Christopher Mason",
"Christos Hatzis",
"Leming Shi",
"Hugo JWL Aerts",
"John Quackenbush"
],
"abstract": "In 2013 we published an analysis demonstrating that drug response data and gene-drug associations reported in two independent large-scale pharmacogenomic screens, Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE), were inconsistent. The GDSC and CCLE investigators recently reported that their respective studies exhibit reasonable agreement and yield similar molecular predictors of drug response, seemingly contradicting our previous findings. Reanalyzing the authors’ published methods and results, we found that their analysis failed to account for variability in the genomic data and more importantly compared different drug sensitivity measures from each study, which substantially deviate from our more stringent consistency assessment. Our comparison of the most updated genomic and pharmacological data from the GDSC and CCLE confirms our published findings that the measures of drug response reported by these two groups are not consistent. We believe that a principled approach to assess the reproducibility of drug sensitivity predictors is necessary before envisioning their translation into clinical settings.",
"keywords": [
"Cancer Cell Lines",
"Pharmacogenomics",
"High-Throughput Screening",
"Biomarkers",
"Drug Response",
"Experimental Design",
"Bioinformatics",
"Statistics"
],
"content": "Introduction\n\nPharmacogenomic studies correlate genomic profiles and sensitivity to drug exposure in a collection of samples to identify molecular predictors of drug response. The success of validation of such predictors depends on the level of noise both in the pharmacological and genomic data. The groundbreaking release of the Genomics of Drug Sensitivity in Cancer1 (GDSC) and Cancer Cell Line Encyclopedia2 (CCLE) datasets enables the assessment of pharmacogenomic data consistency, a necessary requirement for developing robust drug sensitivity predictors. Below we briefly describe the fundamental analytical differences between our initial comparative study3 and the recent assessment of pharmacogenomic agreement published by the GDSC and CCLE investigators4.\n\nThe first GDSC and CCLE studies were published in 2012 and the investigators of both studies have continued to generate data and to release them publicly. One would imagine that any comparative study would use the most current versions of the data. However, the authors of the reanalysis used an old release of the GDSC (July 2012) and CCLE (February 2012) pharmacological data, resulting in the use of outdated IC50 values, as well as missing approximately 400 new drug sensitivity measurements for the 15 drugs screened both in GDSC and CCLE. Assessing data that are three years old and which have been replaced by the very same authors with more recent data seems to be a substantial missed opportunity. It raises the question as to whether the current data would be considered to be in agreement and which data should be used for further analysis.\n\nGiven the complexity and high dimensionality of pharmacogenomic data, the development of drug sensitivity predictors is prone to overfitting and requires careful validation. In this context, one would expect the most significant predictors derived in GDSC to accurately predict drug response in CCLE and vice versa. This will be the case if both studies independently produce consistent measures of both genomic profiles and drug response for each cell line. In our comparative study3, we made direct comparison of the same measurements generated independently in both studies by taking into account the noise in both the genomic and pharmacological data (Figure 1a). By investigating the authors’ code and methods, we identified key shortcomings in their analysis protocol, which have contributed to the authors’ assertion of consistency between drug sensitivity predictors derived from GDSC and CCLE.\n\n(a) Analysis design used in our comparative study (Haibe-kains et al., Nature 2013) where each data generated by GDSC and CCLE are independently compared to avoid information leak and biased assessment of consistency. (b) Analysis design used by the GDSC and CCLE investigators for their ANOVA analysis where the mutation data generated with GDSC were duplicated for use in the CCLE study. (c) Analysis design for the ElasticNet analysis where the molecular profiles from CCLE were duplicated in the GDSC study and the GDSC IC50 were compared to CCLE AUC data. Differences between our analysis design and those used by the GDSC and CCLE investigators are indicated by yellow signs with exclamation mark symbol.\n\nFor their ANOVA analyses, the authors used drug activity area (1-AUC) values independently generated in GDSC and CCLE, but used the same GDSC mutation data across the two different datasets (Figure 1b; see Methods). By using the same mutation calls for both GDSC and CCLE, the authors have disregarded the noise in the molecular profiles, while creating an information leak between the two studies. For their ElasticNet analysis, the authors followed a similar design by reusing the CCLE genomic data across the two datasets, but comparing different drug sensitivity measures that are IC50 in GDSC vs. AUC in CCLE (Figure 1c; see Methods).\n\nWe are puzzled by the seemingly arbitrary choices of analytical design made by the authors, which raises the question as to whether the use of different genomic data and drug sensitivity measures would yield the same level of agreement. Moreover, by ignoring the (inevitable) noise and biological variation in the genomic data, the authors’ analyses is likely to yield over-optimistic estimates of data consistency, as opposed to our more stringent analysis design3.\n\nIn examining correlation, there is no universally accepted standard for what constitutes agreement. However, the FDA/MAQC consortium guidelines define good correlation for inter-laboratory reproducibility5–8 to be ≥0.8. The authors of the present study used two measures of correlation, Pearson correlation (ρ) and Cohen’s kappa (κ) coefficients, but never clearly defined a priori thresholds for consistency, instead referring to ρ>0.5 as “reasonable consistency” in their discussion. Of the 15 drugs that were compared, their analysis found only two (13%) with ρ>0.6 for AUC and three (20%) above that threshold for IC50. This raises the question whether ρ~0.5–0.6 for one third of the compared drugs should be considered as “good agreement.” If one applies the FDA/MAQC criterion, only one drug (nilotinib) passes the threshold for consistency.\n\nSimilarly, the authors referred to the results of their new Waterfall analysis as reflective of “high consistency,” even though only 40% of drugs had a κ≥0.4, with five drugs yielding moderate agreement and only one drug (lapatinib) yielding substantial agreement according to the accepted standards9. Based on these results, the authors concluded that 67% of the evaluable compounds showed reasonable pharmacological agreement, which is misleading as only 8/15 (53%) and 6/15 (40%) drugs yielded ρ>0.5 for IC50 and AUC, respectively. Taking the union of consistency tests is bad practice; adding more sensitivity measures (even at random) would ultimately bring the union to 100% without providing objective evidence of actual data agreement.\n\nThe authors acknowledged that the consistency of pharmacological data is not perfect due to the methodological differences between protocols used by CCLE and GDSC, further stating that standardization will certainly improve correlation metrics. To test this important assertion, the authors could have analyzed the replicated experiments performed by the GDSC using identical protocols to screen camptothecin and AZD6482 against the same panel of cell lines at the Wellcome Trust Sanger Institute and the Massachusetts General Hospital.\n\nOur re-analyses3,10 of drug sensitivity data from these drugs found a correlation between GDSC sites on par with the correlations observed between GDSC and CCLE (ρ=0.57 and 0.39 for camptothecin and AZD6482, respectively; Figure 2 a,b). These results suggest that intrinsic technical and biological noise of pharmacological assays is likely to play a major role in the lack of reproducibility observed in high-throughput pharmacogenomic studies, which cannot be attributed solely to the use of different experimental protocols.\n\n(a) Camptothecin and (b) AZD6482. PCC: Pearson correlation coefficient; MGH: Massachusetts General Hospital (Boston, MA, USA); WTSI: Wellcome Trust Sanger Institute (Hinxton, UK).\n\nIn their comparative study, the authors did not assess the consistency of genomic data between GDSC and CCLE4. Consistency of gene copy number and expression data were significantly higher than for drug sensitivity data (one-sided Wilcoxon rank sum test p-value=3×10-5; Figure 3), while mutation data exhibited poor consistency as reported previously11. The very high consistency of copy number data is quite remarkable (Figure 3a) and could be partly attributed to the fact that CCLE investigators used their SNP array data to compare cell line fingerprints with those of the GDSC project prior to publication and removed the discordant cases from their dataset2.\n\n(a) Continuous values for gene copy number ratio (CNV), gene expression (EXPRESSION), AUC and IC50 and (b) for binary values for presence/absence of mutations (MUTATION) and insensitive/sensitive calls based on AUC >= 0.2 and IC50 > 1 microMolar values. PCC: Pearson correlation coefficient; Kappa: Cohen's Kappa coefficient.\n\n\nConclusions\n\nWe agree with the authors that their and our observations “[…] raise important questions for the field about how best to perform comparisons of large-scale data sets, evaluate the robustness of such studies, and interpret their analytical outputs.” We believe that a principled approach using objective measures of consistency and an appropriate analysis strategy for assessing the independent datasets is essential. An investigation of both the methods described in the manuscript and the software code used by the authors to perform their analysis4 identified fundamental differences in analysis design compared to our previous published study3. By taking into account variations in both the pharmacological and genomic data, our assessment of pharmacogenomic agreement is more stringent and closer to the translation of drug sensitivity predictors in preclinical and clinical settings, where zero-noise genomic information cannot be expected.\n\nOur stringent re-analysis of the most updated data from the GDSC and CCLE confirms our 2013 finding that the measures of drug response reported by these two groups are not consistent and have not improved substantially as the groups have continued generating data since 201210. While the authors make arguments suggesting consistency, it is difficult to imagine using these post hoc methods to drive discovery or precision medicine applications.\n\nThe observed inconsistency between early microarray gene expression studies served as a rallying cry for the field, leading to an improvement and standardization of experimental and analytical protocols, resulting in the agreement we see between studies published today. We are looking forward to the establishment of new standards for large-scale pharmacogenomic studies to realize the full potential of these valuable data for precision medicine.\n\n\nMethods\n\nThe authors’ software source code. As the authors’ source code, we refer to the ‘CCLE.GDSC.compare’ (version 1.0.4 from December 18, 2015) and DRANOVA (version 1.0 from October 21, 2014) R packages available from http://www.broadinstitute.org/ccle/Rpackage/.\n\nAs evidenced in the authors' code (lines 20 and 29 of CCLE.GDSC.compare::PreprocessData.R), they used GDSC and CCLE pharmacological data released on July 2012 and February 2012, respectively. However the GDSC released updated sets of pharmacological data (release 5) on June 2014, gene expression arrays (E-MTAB-3610) and SNP arrays (EGAD00001001039) on July 2015. CCLE released updated pharmacological data on February 2015, the mutation and SNP array on October 2012, and the gene expression data, on March 2013. These updates substantially increased the overlap in genomic features between the two studies, thus providing new opportunities to investigate the consistency between GDSC and CCLE10.\n\nIn the authors’ ANOVA analyses, identical mutation data were used for both GDSC and CCLE studies as can be seen in the authors’ analysis code in lines 20, 25–35 of CCLE.GDSC.compare::plotFig2A_biomarkers.R.\n\nIn their EN analyses, the authors compared different drug sensitivity measures, using IC50 in GDSC and AUC in CCLE, as described in the Supplementary Data 5 and stated in the Methods section of their published study:\n\n“Since the IC50 is not reported in CCLE when it exceeds the tested range of 8 μM, we used the activity area for the regression as in the original CCLE publication. We also used the values considered to be the best in the original GDSC study: the interpolated log(IC50) values.”\n\nThis was confirmed by looking at the authors’ analysis code, lines 83 and 102 of CCLE.GDSC.compare::ENcode/prepData.R. Moreover, identical genomic data were used for both GDSC and CCLE studies, as described the Methods section of the published study:\n\n“In order to compare features between the two studies, we used the same genomic data set (CCLE).”\n\nThis was confirmed by looking at the authors’ analysis code, lines 17, 38, 51, and 70 of CCLE.GDSC.compare::ENcode/genomic.data.R, and lines 10-11 of CCLE.GDSC.compare::plotFigS6_ENFeatureVsExpected.R.\n\nAll analyses were performed using the most updated version of the GDSC and CCLE pharmacogenomic data based on our PharmacoGx package12 (version 1.1.4).\n\nAll analyses were performed using the most updated version of the GDSC and CCLE pharmacogenomic data based on our PharmacoGx package12 (version 1.1.4). PharmacoGx provides intuitive function to download, intersect and compare large pharmacogenomics datasets. The PharmacoSet for the GDSC and CCLE datasets are available from pmgenomics.ca/bhklab/sites/default/files/downloads/ using the downloadPSet() function. The code and the data used to generate all the results and figures are available as Data Files 1 and 2. The code is also available on GitHub: github.com/bhklab/cdrug-rebuttal.\n\nIn the Methods, the authors use all cell lines to optimally identify the inflection point in the response distribution curves. The authors stated that “This is a major difference to the Haibe-Kains et al. analysis, as that analysis only considered the cell-lines in common between the studies when generating response distribution curves.” This is not correct. As can be seen in our publicly available R code, we performed the sensitivity calling (using the Waterfall approach as published in the CCLE study2 before restricting our analysis to the common cell lines, for the obvious reasons that the authors mentioned in their manuscript. See lines 308 and 424 in https://github.com/bhklab/cdrug/blob/master/CDRUG_format.R.\n\n\nData and software availability\n\nOpen Science Framework: Dataset: Assessment of pharmacogenomic agreement, doi 10.17605/osf.io/47rfh13",
"appendix": "Author contributions\n\n\n\nZ Safikhani, N El-Hachem R Quevedo, and P Smirnov were responsible for downloading and curating the pharmacogenomic data. Z Safikhnao wrote most of the analysis code with the help of N El-Hachem R Quevedo, and P Smirnov. Z Safikhani, J Quackenbush and B Haibe-Kains designed the study. B Haibe-Kains supervised the study. All authors participated in the interpretation of the results. Z Safikhani, A Goldenberg, N Juul Birkbak, C Mason, C Hatzis, L Shi, H Aerts, J Quackenbush and B Haibe-Kains participated in the manuscript writing.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nZ Safikhani was supported by the Cancer Research Society (Canada; grant #19271) and the Ontario Institute for Cancer Research through funding provided by the Government of Ontario. P Smirnov was supported by the Canadian Cancer Society Research Institute. C Hatzis was supported by Yale University. N Juul Birkbak was funded by The Villum Kann Rasmussen Foundation. C Mason was supported by the Starr Cancer Consortium grants (I7-A765, I9-A9-071), Irma T. Hirschl and Monique Weill-Caulier Charitable Trusts, Bert L and N Kuggie Vallee Foundation, WorldQuant Foundation (CEM), Pershing Square Sohn Cancer Research Alliance, NASA (NNX14AH50G), and the National Institutes of Health (R25EB020393, R01NS076465). L Shi was supported by the National High Technology Research and Development Program of China (2015AA020104), the National Natural Science Foundation of China (31471239), the 111 Project (B13016), and the National Supercomputer Center in Guangzhou, China. J Quackenbush was supported by grants from the NCI GAME-ON Cancer Post-GWAS initiative (5U19 CA148065) and the NHLBI (5R01HL111759). B Haibe-Kains was supported by the Gattuso Slaight Personalized Cancer Medicine Fund at Princess Margaret Cancer Centre.\n\n\nAcknowledgements\n\nThe authors would like to thank the investigators of the Genomics of Drug Sensitivity in Cancer (GDSC) and the Cancer Cell Line Encyclopedia (CCLE) who have made their invaluable data available to the scientific community. We thank the MAQC/SEQC consortium for their constructive feedback.\n\n\nReferences\n\nGarnett MJ, Edelman EJ, Heidorn SJ, et al.: Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature. 2012; 483(7391): 570–575. 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): 603–607. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaibe-Kains B, El-Hachem N, Birkbak NJ, et al.: Inconsistency in large pharmacogenomic studies. Nature. 2013; 504(7480): 389–393. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCancer Cell Line Encyclopedia Consortium, Genomics of Drug Sensitivity in Cancer Consortium: Pharmacogenomic agreement between two cancer cell line data sets. Nature. 2015; 528(7580): 84–87. PubMed Abstract | Publisher Full Text\n\nSEQC/MAQC-III Consortium: A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequencing Quality Control Consortium. Nat Biotechnol. 2014; 32(9): 903–914. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMAQC Consortium, Shi L, Reid LH, et al.: The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. Nat Biotechnol. 2006; 24(9): 1151–1161. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShi L, Campbell G, Jones WD, et al.: The MicroArray Quality Control (MAQC)-II study of common practices for the development and validation of microarray-based predictive models. Nat Biotechnol. 2010; 28(8): 827–838. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi S, Tighe SW, Nicolet CM, et al.: Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study. Nat Biotechnol. 2014; 32(9): 915–925. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSim J, Wright CC: The kappa statistic in reliability studies: use, interpretation, and sample size requirements. Phys Ther. 2005; 85(3): 257–268. PubMed Abstract\n\nSafikhani Z, Freeman M, Smirnov P, et al.: Revisiting inconsistency in large pharmacogenomic studies. bioRxiv. 2015; 026153. Publisher Full Text\n\nHudson AM, Yates T, Li Y, et al.: Discrepancies in cancer genomic sequencing highlight opportunities for driver mutation discovery. Cancer Res. 2014; 74(22): 6390–6396. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmirnov P, Safikhani Z, El-Hachem N, et al.: PharmacoGx: An R package for analysis of large pharmacogenomic datasets. Bioinformatics. 2015; 32(8): 1244–1246. pii: btv723. PubMed Abstract | Publisher Full Text\n\nSafikhani Z, El-Hachem N, Quevedo R, et al.: Dataset: Assessment of pharmacogenomic agreement. Open Science Framework. 2016. Data Source"
}
|
[
{
"id": "14316",
"date": "10 Jun 2016",
"name": "Weida Tong",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 lot to take/digest the manuscript. I break this story into three parts:\nIn 2012, both GDSC and CCLE released/published drug sensitivity data (both pharmacological and genomic). In 2013, the authors compared the two studies using the drugs in common between two. Their analysis was carried out in a direct fashion which account for variations of both genomic and pharmacological data from the same site (GDSC or CCLE) and found the results between two did not agree.\n\nRecently, GDSC/CCLE did an independent analysis and demonstrated that the agreement between two are actually higher (using ANOVA) than what the authors reported. They concluded that the results between GDSC and CCLE were consistent. However, the comparison was only focused on the pharmacological data because the genomic data used actually came from one site. That means their analysis did not include the noise introduced by both sites in this comparison.\n\nThe authors, again, reanalyzed data by including pharmacological and genomic data from both sites and the conclusions remain as the same as they reported in 2013.\nI have no problem with their analysis and support their conclusions. With that said, I did find the paper could flow better by moving two sections into Discussion. These are:\n“Which pharmacological drug response data should one use?” - It seems odd and smell bad that GDSC/CCLE used the data published in 2012 and totally ignored the most current data in their analysis. This could be due to many different reasons. Thus, speculation is not necessary considered as “results”. I would say this will be better justified as “discussion”.\n\n“What constitutes agreement” – Again, this is a difficult call. I believe there is no single baseline that can be used to justify consistency. Thus, most text in this section will sit better in “discussion”.\nOverall, I support its indexation with revision by focusing on the flow of the story and the structure of manuscript.",
"responses": []
},
{
"id": "14596",
"date": "27 Jun 2016",
"name": "Terence Speed",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI found the title appropriate, and that the abstract represented a suitable summary of the work.\n\nI believe that the design, methods and analysis of results are appropriate for the topic being studied, and that for the most part, they were clearly explained. A couple of perceived shortcomings are itemized here.\n\np.3, column 2, line 2. The “but” would be better replaced by “and”.\n\np.5. Figure 2. The dotted and solid diagonal lines on these plots are not identified in either the caption or the text.\n\np.5, Figure 3. It is nowhere explained whose Pearson correlations (PCC) are summarized in these box plots. I suppose that some number (to be stated) of cell lines were profiled in both GDSC and CCLE, and that in all cases, the PCC in the box plots are calculated from molecular data from pairs consisting of the data on the same cell line generated in GDSC and in CCLE. A clear statement along these lines would be helpful.\n\np.6, column 1, lines 1-4. This assertion would have more force if the authors told the reader how many cell lines could have contributed PCC to the box plot of Figure 3a, and how many did do so.\n\nFurther, I do believe that the conclusions are sensible, balanced and justified on the basis of the results of the study.\n\nFinally, I understand that all the data used in this study is available, and this is also true for the code used to generate all the results and figures.",
"responses": []
},
{
"id": "14152",
"date": "29 Jun 2016",
"name": "Yudi Pawitan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper highlights the curious lack of rigorous standards for what constitutes ‘agreement’, ‘consistency’ between genomic studies, or more generally, the fundamental issues of ‘validation’ and ‘reproducibility’, etc. The problem is even more serious of results based on high-throughput omics data as the potential for false positive is substantial.\n\nThe persistent lack of consensus or standards may partly indicate that these issues are not so straightforward. The main problem is that when we say we ‘validate’ a result, this can be done at different strengths. For example, consider the commonly performed method in statistical analyses, the so-called ‘cross-validation’, where we split our total sample into training and validation sets. If the split is done randomly, then we have only a ‘soft validation’, since it applies to the same sample (or same lab, same population, same measurement method, etc) so the ‘validation’ is internal and corresponds to statistical significance only. In contrast a scientist may wish for something stronger, for an external validation, for example, for the ‘biological truth’ to apply other populations; thus, one study may be performed in a European population, but the external validation is done in an Asian population. The latter is a stronger validation than the random-split validation, giving a more compelling and general biological story. What is relevant here is that both validations are commonly done in practice, and both are valid, but they carry different levels of information. I think what matters in practice is that the implication of the validation should always be clear (or clarified), so that the user of the information can judge its relevance.\n\nThe key point of Safikhani et al is that their 2013 validation study of the genomic predictors of drug-sensitivity was more stringent than the 2015 validation studies by the GDSC and CCLE investigators. This is clearly highlighted in Figure 1, where the latter used the same molecular data, so the ‘validation’ is only of the pharmacological data and perhaps (not clear to me) the method of analyses. Which level of validation is more relevant here? Let us imagine how the results (eg the genomic predictors) are to be used in patients. The molecular data are likely to be generated and analyzed in a diversity of labs, so the genomic predictors should really be robust to the actual heterogeneity in the molecular data. The results (the genomic predictors) may not survive such stringent requirements, but that is what we need to know. So, overall, I agree with Safikhani et al that a more stringent validation allowing for variability in both molecular and pharmacological data is more relevant in this context of drug prediction.\n\n(However, reading Haibe-Kains et al, there seemed to be an emphasis that the failure of agreement was due to the high variability in the pharmacological data. So it is possible that the later studies by the GDSC-CCLE investigators responded to this concern only.)\nRegarding specific issues in the paper:\nI do not consider the use of most recent data as a key issue. I agree that the choice of IC50 in GDSC vs AUC in CCLE is puzzling and only raises a question mark regarding the results. Arbitrary cutoffs in defining what constitutes an ‘agreement’ are unnecessary if authors can refrain from using judgmental words like ‘high consistency’ etc., especially when used as a summary statement across distinct drugs. It would be better to just report the actual performance for each drug or for each cancer type, since it is still not clear how these statistics would translate in terms of clinical cost-benefit balance.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-825
|
https://f1000research.com/articles/5-803/v1
|
05 May 16
|
{
"type": "Review",
"title": "Current clinical immunotherapeutic approaches for head and neck cancer",
"authors": [
"Carolina Soto Chervin",
"Bruce Brockstein",
"Carolina Soto Chervin"
],
"abstract": "It was estimated that 59,340 new cases of head and neck cancer would be diagnosed in the US alone in 2015 and that 12,290 deaths would be attributed to the disease. Local and regional recurrences may be treated with chemotherapy and radiation; however, metastatic head and neck cancer is fatal and is treated with chemotherapy for palliation. Recent successful treatment of a variety of solid and hematological malignancies by immunotherapeutic approaches (i.e. harnessing the body’s own immune system to combat disease) has added a fourth therapeutic option for the treatment of cancer. This commentary will review the status of immunotherapies in clinical development for the specific treatment of head and neck cancer.",
"keywords": [
"Autologous Cell Therapy",
"tumor-infiltrating lymphocytes",
"Immune Checkpoint Inhibitors",
"DNA-based vaccine",
"Chimeric antigen receptors"
],
"content": "Introduction\n\nSquamous cell cancers of the head and neck, including the oral cavity, pharynx, and larynx, together account for the eighth most common malignancy in the US and represent an even higher percentage worldwide. In 2015, a total of 59,340 new cases and 12,290 estimated deaths were expected1. Despite a decrease in the number of smoking-related cases of head and neck squamous cell cancers (HNSCC), the overall incidence is steady or increased because of the epidemic of squamous cell cancers of the oropharynx attributable to human papillomavirus (HPV).\n\nAlthough advances in local therapies have led to improved survival for locoregionally advanced HNSCC, fatal local recurrence and metastases remain a very significant problem. Only small advances have been made in survival rates or survival duration with various chemotherapies in the last several decades. Even with modern multi-agent chemotherapy, the expected median survival for a patient with an incurable or metastatic relapse remains under a year, or marginally longer for patients who develop metastases from an HPV-related HNSCC2,3. Improvements in systemic therapy are needed for this group of otherwise incurable patients. In addition, as systemic therapy plays a key role in the multi-modality curative intent therapy for locoregionally advanced HNSCC, better systemic therapy is needed to increase cure rates in patients with locoregionally advanced but not metastatic HNSCC.\n\nImmunotherapy has been tested in the past in HNSCC. Trials with systemic interferon-alpha (IFN-α) or interleukin-2 (IL-2) were disappointing, and response rates ranged from 0 to 6%. One trial examining IFN-α in combination with IL-2 showed only a modest effect; two of 11 patients (18%) exhibited a partial response4. Given the relative lack of success of these trials and others, and the significant toxicity of these drugs, new therapeutic approaches were needed4–10.\n\nStandard-of-care cancer treatment has revolved around three traditional therapies: radiation, chemotherapy, and surgery. With the advent of immunotherapy, the promise of harnessing and focusing the body’s own immune system to combat malignancies has added a powerful weapon to the oncologist’s arsenal. The remainder of this review will focus on currently approved immunotherapy as well as promising immunotherapeutic approaches under clinical investigation.\n\n\nVaccines\n\nOne of the most heavily investigated areas of cancer immunotherapy has centered on vaccines. They have been applied in most cancer indications and their lack of success has been well documented. However, there continue to be trials with new adjuvants combined with different vaccination components (e.g., peptide, DNA, and bacteria). Within head and neck cancer, a DNA-based vaccine targeting E6 and E7 genes of HPV in combination with IL-12 is currently under investigation in a phase 1 clinical trial (NCT02163057). Recent work with immunomodulatory peptide vaccines for HPV-positive and MAGE-A3-positive tumors elicited antigen-specific T cell and antibody responses to the respective vaccines but ultimately lacked clinical efficacy (NCT00257738)11. Another study incorporated personalized medicine into vaccine therapy in a phase I/II trial investigating the safety profile of a vaccine composed of patient-specific tumor antigen derived from chaperone-rich tumor cell lysate (NCT01998542). A phase I trial was recently completed by using a dendritic cell p53 vaccine, but further studies need to be conducted in order to gauge clinical efficacy12.\n\n\nAutologous cell therapy: tumor infiltrating lymphocytes\n\nAdoptive cell therapy involves the transfer of tumor-reactive T cells, cultured ex vivo, into patients. With improvement in cell culturing (e.g., IL-2 conditioning), these T cells, which traditionally have been isolated from tumors (tumor-infiltrating lymphocytes, or TILs), could be cultured ex vivo, generating a sufficient number of cells before re-introduction into the patient. This therapeutic regimen was pioneered in the field of melanoma and is still employed experimentally for the treatment of various solid tumors, including HNSCC13,14. For HPV-associated HNSCC, a phase 2 clinical trial under way at the National Cancer Institute is generating TILs from metastatic HPV16/HPV18-positive tumors (NCT01585428). Undifferentiated nasopharyngeal cancers have a high association with Epstein-Barr virus (EBV) and can be treated by using EBV-specific T cells. Unlike TIL preparation, EBV-specific T cells can be isolated and expanded from peripheral blood mononuclear cells (PBMCs). Infusion of EBV-specific T cells showed a durable clinical response in four of six patients who had been refractory to chemotherapy and radiation15. Recent results from a phase 2 trial of EBV-specific autologous T cells in combination with gemcitabine and carboplatin showed a 71% response rate (35 patients), but the 3-year survival rate was 37%16. Currently, two clinical trials using autologous EBV-specific T cells are under way: a phase 2 trial using EBV-specific T cells as a monotherapy (NCT00431210) and a separate phase 3 trial using EBV-specific T cells in combination with gemcitabine and carboplatin versus gemcitabine or carboplatin alone (NCT02578641).\n\n\nAutologous cell therapy: chimeric antigen receptor T cell\n\nIn recent years, adoptive cell therapy has incorporated gene transfer techniques to endow T cells with receptors specific for tumor antigens. This allows the use of a naïve (with regard to antigen specificity) T cell population—a population isolated from patients’ peripheral blood rather than from a resected tumor. Chimeric antigen receptors (CARs) are composed of antibody variable domains specific for a cell surface antigen fused to intracellular T cell co-stimulatory and activation domains (4-1BB and CD3 are most commonly used). When introduced into T cells, these chimeric receptors allow the recognition and subsequent activation against a non-human leukocyte antigen (non-HLA) restricted tumor antigen. Recently, much fanfare has been focused on the use of CARs in hematological malignancies because of seminal work by Carl June and colleagues in chronic lymphocytic leukemia17–19. However, the question remains whether the successes in treating hematological cancers can be transferred to solid tumors owing not the least to the suppressive tumor microenvironment against T cells indicative of many solid tumor types (e.g., upregulation of inhibitory receptors on T cells)20. Within HNSCC, there is currently an ongoing phase 1 trial using an ErbB-specific CAR21,22 (NCT01818323). Engineered T cells expressing the T1E28z CAR, an ErbB ligand fused to CD28 and CD3, will be delivered directly by injection into tumors of HNSCC patients with locoregional disease22. Preclinical data of HNSCC CAR-T therapy suggest that this approach could have a therapeutic effect. A CAR recognizing the EBV latent membrane protein 1 (LMP1) exhibited specific cytolytic activity against LMP1-positive nasopharyngeal tumor line and reduced tumor growth in a xenograft model23. A separate study of a CAR targeting chondroitin sulfate proteoglycan 4 (CSPG4) showed reduced tumor growth in a xenograft model of the HNSCC cell line PCI-3024.\n\n\nImmune checkpoint inhibitors\n\nImmunological responses are carefully regulated through calculated expression of both stimulatory and inhibitory signals. Immune checkpoint receptors provide an example of inhibitory regulation of adaptive immune responses. Their expression plays an essential role in controlling and reducing tissue damage due to robust immune responses during an infection or as a result of self-directed immunity. Programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) are two membrane proteins expressed by T cells that have been identified as key inhibitory surface receptors in T cell function (Figure 1). Their activation is associated with exhaustive phenotypes in tumor-infiltrating CD3+ T cells. Tumors have evolved mechanisms to successfully reduce the anti-tumor response of T cells via induction of such immune checkpoint inhibitors. As such, blockade of these receptors or their ligands is an attractive approach to promote a T cell-directed immune response against the tumor25.\n\nT cell activation is influenced by co-stimulatory signals received via CD28. Conversely, T cell activity can be reduced by upregulation of inhibitory receptors programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4).\n\nNivolumab is an anti-PD-1 monoclonal antibody that was recently approved by the US Food and Drug Administration (FDA) for the treatment of advanced metastatic cancers, specifically melanoma, non-small cell lung cancer, and renal cell carcinoma. A phase 3 clinical trial demonstrated improved overall survival with nivolumab treatment compared with docetaxel chemotherapy in patients with non-small cell lung cancer (39% versus 23% at 18 months)26. In patients with HNSCC refractory to platinum therapy, a phase 3 study of nivolumab monotherapy resulted in improved overall survival compared with treatment with the investigator’s choice of weekly methotrexate, docetaxel, or cetuximab (NCT02105636). Ipilimumab, an anti-CTLA-4 monoclonal antibody, was approved for the treatment of metastatic melanoma in 2011 on the basis of a phase 3 trial that compared it with a gp100 peptide vaccine25. Subsequently, two PD-1 inhibitors, nivolumab and pembrolizumab, were approved for second-line use after ipilimumab. Improved progression-free survival was also observed with nivolumab compared with ipilimumab for metastatic melanoma, both as monotherapy and in combination with an additional checkpoint inhibitor, ipilimumab27. Combined, ipilimumab and nivolumab extended progression-free survival by 8.5 and 4.5 months as compared with monotherapy with ipilimumab or nivolumab, respectively, and the FDA recently approved the combination for melanoma in the first-line setting for BRAF wild-type patients or following BRAF resistance. Most importantly, these drugs appear to lead to “cure” in a substantial portion of patients with metastatic melanoma, though less commonly in other cancers. The amazing success of these checkpoint inhibitors has led to a race for approval in other solid tumor indications, including HNSCC. Focusing on PD-1 blockade, or blockade of its ligand PD-L1 in advanced HNSCC, a multitude of trials are under way (Table 1). Recent studies have concentrated on characterizing PD-1/PD-L1 expression and its correlation with overall outcome. HPV-positive HNSCC tumors were shown to have a unique immune profile compared with HPV-negative tumors. They are associated with a higher density of CD8+ T cells, which can be stimulated to produce IFN-γ and IL-17 in vitro28. Although efforts have focused on the profile of PD-1/PD-L1 intra-tumoral expression, there is a lack of data correlating expression in HPV-positive HNSCC with overall survival28–30. In fact, at least one study unexpectedly correlated improved overall outcomes with increased levels of PD-1+ T cells in HPV-positive tumors31.\n\nCTLA-4, cytotoxic T lymphocyte-associated antigen 4; HNSCC, head and neck squamous cell cancer; HPV, human papillomavirus; PD-1, programmed death 1; PD-L1, programmed death-ligand 1.\n\n\nSummary\n\nIt remains to be seen whether adoptive cellular therapy alone will be sufficient to affect a sustainable anti-tumor response in HNSCC. The approvals of checkpoint inhibitor antibodies for melanoma (ipilimumab, nivolumab, and pembrolizumab), non-small cell lung carcinoma (nivolumab and pembrolizumab), and most recently renal cell carcinoma (nivolumab) have paved the way for these foundational therapies; that is, one or more checkpoint inhibitors will likely be incorporated into solid tumor therapeutic regimens. As such, there is an extensive clinical trial program for approval in new indications, including cancers of the head and neck. It is not hard to imagine that the next generation of immunotherapy would combine these checkpoint inhibitors with engineered T cells (e.g., CARs and modified T cell receptors) or even autologous T cells.\n\nHowever, the attractiveness of antibody therapy is offset by the headache of cellular therapy. Whereas checkpoint inhibitors represent classic “off-the-shelf” biopharmaceutical agents, cellular therapy remains “personalized” medicine. From a manufacturing/business standpoint, it is still not yet feasible to mass-produce cellular therapies, although industry is hard at work solving this problem. As such, adoptive therapy approaches are still years away from “off-the-shelf” status32,33.",
"appendix": "Competing interests\n\n\n\nBB is a consultant (on the advisory board for melanoma) for Bristol-Meyers Squibb (New York, NY, USA). CSC declares that she has no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAmerican Cancer Society: Cancer Facts & Figures 2015. Atlanta: American Cancer Society; 2015. Reference Source\n\nFakhry C, Zhang Q, Nguyen-Tan PF, et al.: Human papillomavirus and overall survival after progression of oropharyngeal squamous cell carcinoma. J Clin Oncol. 2014; 32(30): 3365–73. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nArgiris A, Li S, Ghebremichael M, et al.: Prognostic significance of human papillomavirus in recurrent or metastatic head and neck cancer: an analysis of Eastern Cooperative Oncology Group trials. Ann Oncol. 2014; 25(7): 1410–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUrba SG, Forastiere AA, Wolf GT, et al.: Intensive recombinant interleukin-2 and alpha-interferon therapy in patients with advanced head and neck squamous carcinoma. Cancer. 1993; 71(7): 2326–31. PubMed Abstract | Publisher Full Text\n\nVlock DR, Andersen J, Kalish LA, et al.: Phase II trial of interferon-alpha in locally recurrent or metastatic squamous cell carcinoma of the head and neck: immunological and clinical correlates. J Immunother Emphasis Tumor Immunol. 1996; 19(6): 433–42. PubMed Abstract\n\nCascinu S, Del Ferro E, Ligi M, et al.: Phase II trial of 13-cis retinoic acid plus interferon-alpha in advanced squamous cell carcinoma of head and neck, refractory to chemotherapy. Ann Oncol. 1996; 7(5): 538. PubMed Abstract | Publisher Full Text\n\nVoravud N, Lippman SM, Weber RS, et al.: Phase II trial of 13-cis-retinoic acid plus interferon-alpha in recurrent head and neck cancer. Invest New Drugs. 1993; 11(1): 57–60. PubMed Abstract | Publisher Full Text\n\nRoth AD, Abele R, Alberto P: 13-cis-retinoic acid plus interferon-alpha: a phase II clinical study in squamous cell carcinoma of the lung and the head and neck. Oncology. 1994; 51(1): 84–6. PubMed Abstract | Publisher Full Text\n\nChi KH, Myers JN, Chow KC, et al.: Phase II trial of systemic recombinant interleukin-2 in the treatment of refractory nasopharyngeal carcinoma. Oncology. 2001; 60(2): 110–5. PubMed Abstract | Publisher Full Text\n\nMattijssen V, De Mulder PH, De Graeff A, et al.: Intratumoral PEG-interleukin-2 therapy in patients with locoregionally recurrent head and neck squamous-cell carcinoma. Ann Oncol. 1994; 5(10): 957–60. PubMed Abstract\n\nZandberg DP, Rollins S, Goloubeva O, et al.: A phase I dose escalation trial of MAGE-A3- and HPV16-specific peptide immunomodulatory vaccines in patients with recurrent/metastatic (RM) squamous cell carcinoma of the head and neck (SCCHN). Cancer Immunol Immunother. 2015; 64(3): 367–79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchuler PJ, Harasymczuk M, Visus C, et al.: Phase I dendritic cell p53 peptide vaccine for head and neck cancer. Clin Cancer Res. 2014; 20(9): 2433–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRosenberg SA, Lotze MT, Muul LM, et al.: Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med. 1985; 313(23): 1485–92. PubMed Abstract | Publisher Full Text\n\nRosenberg SA, Packard BS, Aebersold PM, et al.: Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report. N Engl J Med. 1988; 319(25): 1676–80. PubMed Abstract | Publisher Full Text\n\nStraathof KC, Bollard CM, Popat U, et al.: Treatment of nasopharyngeal carcinoma with Epstein-Barr virus--specific T lymphocytes. Blood. 2005; 105(5): 1898–904. PubMed Abstract | Publisher Full Text\n\nChia WK, Teo M, Wang WW, et al.: Adoptive T-cell transfer and chemotherapy in the first-line treatment of metastatic and/or locally recurrent nasopharyngeal carcinoma. Mol Ther. 2014; 22(1): 132–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKalos M, Levine BL, Porter DL, et al.: T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med. 2011; 3(95): 95ra73. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPorter DL, Levine BL, Kalos M, et al.: Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011; 365(8): 725–33. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPorter DL, Hwang WT, Frey NV, et al.: Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med. 2015; 7(303): 303ra139. PubMed Abstract | Publisher Full Text\n\nMoon EK, Wang LC, Dolfi DV, et al.: Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clin Cancer Res. 2014; 20(16): 4262–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPapa S, van Schalkwyk M, Maher J: Clinical Evaluation of ErbB-Targeted CAR T-Cells, Following Intracavity Delivery in Patients with ErbB-Expressing Solid Tumors. Methods Mol Biol. 2015; 1317: 365–82. PubMed Abstract | Publisher Full Text\n\nvan Schalkwyk MC, Papa SE, Jeannon JP, et al.: Design of a phase I clinical trial to evaluate intratumoral delivery of ErbB-targeted chimeric antigen receptor T-cells in locally advanced or recurrent head and neck cancer. Hum Gene Ther Clin Dev. 2013; 24(3): 134–42. PubMed Abstract | Publisher Full Text\n\nTang X, Zhou Y, Li W, et al.: T cells expressing a LMP1-specific chimeric antigen receptor mediate antitumor effects against LMP1-positive nasopharyngeal carcinoma cells in vitro and in vivo. J Biomed Res. 2014; 28(6): 468–75. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGeldres C, Savoldo B, Hoyos V, et al.: T lymphocytes redirected against the chondroitin sulfate proteoglycan-4 control the growth of multiple solid tumors both in vitro and in vivo. Clin Cancer Res. 2014; 20(4): 962–71. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHodi FS, O'Day SJ, McDermott DF, et al.: Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010; 363(8): 711–23. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBorghaei H, Paz-Ares L, Horn L, et al.: Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med. 2015; 373(17): 1627–39. PubMed Abstract | Publisher Full Text\n\nLarkin J, Chiarion-Sileni V, Gonzalez R, et al.: Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 2015; 373(1): 23–34. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPartlová S, Bouček J, Kloudová K, et al.: Distinct patterns of intratumoral immune cell infiltrates in patients with HPV-associated compared to non-virally induced head and neck squamous cell carcinoma. Oncoimmunology. 2015; 4(1): e965570. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLyford-Pike S, Peng S, Young GD, et al.: Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPV-associated head and neck squamous cell carcinoma. Cancer Res. 2013; 73(6): 1733–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim HS, Lee JY, Lim SH, et al.: Association Between PD-L1 and HPV Status and the Prognostic Value of PD-L1 in Oropharyngeal Squamous Cell Carcinoma. Cancer Res Treat. 2016; 48(2): 527–36. PubMed Abstract | Publisher Full Text\n\nBadoual C, Hans S, Merillon N, et al.: PD-1-expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancer. Cancer Res. 2013; 73(1): 128–38. PubMed Abstract | Publisher Full Text\n\nKaiser AD, Assenmacher M, Schröder B, et al.: Towards a commercial process for the manufacture of genetically modified T cells for therapy. Cancer Gene Ther. 2015; 22(2): 72–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCooper LJ: Moving from tinkering in the garage to assembly line production: the manufacture of genetically modified T cells expressing chimeric antigen receptors (CARs) comes on line. Cancer Gene Ther. 2015; 22(2): 64–6. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13716",
"date": "05 May 2016",
"name": "Francis Worden",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13717",
"date": "05 May 2016",
"name": "Rainald Knecht",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-803
|
https://f1000research.com/articles/5-802/v1
|
05 May 16
|
{
"type": "Systematic Review",
"title": "Socio-cultural aspects of gender-based violence and its impacts on women’s health in South Asia",
"authors": [
"Ghose Bishwajit",
"Sajeeb Sarker",
"Sanni Yaya",
"Sajeeb Sarker",
"Sanni Yaya"
],
"abstract": "This review attempts to shed light on the socio-cultural roots of gender-based violence (GBV) and its impact on women's health with a special reference to reproductive health in the context of South Asia. It also identifies the policy and capacity gaps that impede the implementation of gender-related development goals and makes recommendations in light of the ongoing situation.\n\nLed by the growing recognition of the pivotal importance of women’s and child’s health in national development processes, the issues surrounding gender-based violence (GBV) are being given increasing prominence in the global public health agenda. However, developing regions such as South Asia and Sub Saharan Africa are lagging far behind in this respect and failing to prioritize and implement gender-related development strategies. South Asian nations in their pursuance of gender-related goals are faced with host of infrastructure issues in financing, policy guidance, implementation and legislation terms. This study highlights the fact that GBV is essentially a socio-cultural issue which calls for developing gender-sensitive social policies and making strategic investment to promote social capital tailored especially to promote a more nuanced view of women’s health and human rights. Method: Cochrane Database of Systematic Reviews, Embase, Ovid MEDLINE, PsycINFO, and Web of Science were searched for original and review articles published between January of 2000 to July of 2015. Boolean search was performed to identify suitable articles relating to GBV conducted on South Asia (Bangladesh, India, Nepal, Pakistan, Sri Lanka) by using the following search terms: South Asia, GBV, IPV (intimate partner violence), domestic violence, women’s health, reproductive health, risk factors, perpetrator, sexual abuse. Reference lists were searched manually for articles relevant to this study (snowballing). One volunteer from each country included in the study helped in reviewing renowned local media reports and constitutions to gather policy information germane to GBV issues.",
"keywords": [
"GBV",
"IPV",
"Human right",
"South Asia",
"Healthcare",
"Women’s health"
],
"content": "Introduction\n\nDespite the widespread recognition of the critical importance of women’s health in the process of socio-economic progress, a confluence of socio-political factors continue to thwart the efforts to advance women’s health status and human rights. Globally, gender-based violence (GBV) remain the largest single cause of morbidity and mortality among women aged between 15–49 years, claiming more lives than cancer, malaria, traffic accident and war combined (Murthy, 2009). Women’s health issues began to attract special focus in the arena of global public health since the 1990s with GBV coming into spotlight with the publication of the United Nations Declaration on the Elimination of Violence against Women (DEVAW) in 1993.\n\nGBV is regarded as a significant public health issue and is a major risk factor of women’s health related vulnerabilities (mainly reproductive and psychological health), especially in the underdeveloped regions like South Asia. This study explores the socio-cultural roots of gender gradient and the impact of GBV on women’s reproductive health in this region. Violence against women (VAW) is observed in all societies both industrialized and developing. In South Asia, however, the violence statistics depicts a heart-rending scenario (described in the third section). Prevalence of IPV (Intimate partner violence) is strikingly high in South Asia, particularly with regard to spousal violence, early marriages, honor-killing (HK), forced sex and sex trafficking (WHO, 2005). In Bangladesh, a country of around 160 million people where the incidence of VAW is deplorably high (Johnston & Naved, 2008), the first country representative survey on gender violence was conducted only once in 2011.\n\nA recent World Bank report titled ‘Violence against Women and Girls Violence against Women and Girls: Lessons from South Asia Lessons from South Asia’ presents the most comprehensive GBV issues in south Asia. The report highlights that: 1) South Asia has the highest female to male child mortality rate than anywhere else in the world and 2) has the highest rate of child marriage with remarkably high incidence of intimate partner violence (IPV) (Solotaroff & Prabha, 2014). Child marriage together with dowry, and illiteracy constitute the most common set of risk factors of domestic violence in South Asia. Data from a nationally representative study in Pakistan showed that in about half of the marriages, the women were less than 18 years of age. In Nepal, 40% of girls get married by the age of 15 with 7% before reaching 10 (Nasrullah et al., 2009). In India, where 68% of the female population is illiterate, 47.4% of girls aged between 20–24 years got married before 18, and 42% become mother before reaching 20 (NFHS-3, 2005–06). Governments’ commitment to address gender-related crimes are reflected through various policies: The Dowry Prohibition Act (1980; 1986) and The Prevention of Women and Child Repression Act (2000) in Bangladesh; The Dowry Prohibition Act (1961, Amended in 1986) and Protection of Women from Domestic Violence Act (2005) in India; Domestic Violence Act (2009) in Nepal, Women Protection Act (2006) in Pakistan, The Prevention of Domestic Violence Act (PDVA, 2005) in Sri Lanka. Although governments and civil society organizations (CSOs) are constantly striving to curb gender related crimes (Naved & Akhtar, 2008), lack of comprehensive data and failure to detect and address the underlying causes stemming from various socio-cultural complexities are hampering progress. As described in the following section, GBV is fundamentally a by-product of the complex interplay of religious, ideological, socio-cultural factors. Entanglement of GBV with traditional socio-cultural dimensions is the main reason legislative interventions alone have had little impact. This culturally intricate nature of the problem calls for developing socially innovative programs tailored to encourage ideological changes and embracing more ethically acceptable norms. As a potential solution to this intricate problem, previous studies have recognized the need for developing a more gender-sensitive policy framework by aligning the various socio-political, cultural and economic factors that underpin health and human rights of women (Johnston & Naved, 2008). This means there is a strong need for policy makers to adopt more timely decisions and innovative strategies by increasing cooperation with representatives of civil societies and private sectors, local and international human rights non-governmental organizations (NGOs) and donor agencies (Actually I meant organization which operate in a political context and have commitments to donate. Charities act occasionally, but donor agencies act more routinely when needs arise) in the effort to effectively address gender-related issues.\n\n\nWomen’s attitude to domestic violence: the south Asian context\n\nThough globalization has brought about certain changes in social values and attitudes towards gender issues in South Asia, traditional patriarchal values still persist in many societies and contribute to the erosion of women’s human rights. Stringent socio-cultural practices and patriarchal attitudes are reported to give rise to devaluation of the role of women and increase the likelihood VAW (Niaz, 2003). In certain communities or social segments, arranged marriage, dowry, and restriction of female socialization to some degree are considered supportive of preserving the familial norms and values that has been passed down through generations. Paradoxically, though this segment is getting increasingly smaller in number and has been practicing the traditional way without experiencing any noticeably adverse impacts in the personal and/or social sphere, it silently imparts the dominant patriarchal social system, which renders the complete abolition or prevention of the traditional gender-biased practices an extremely complex task for legal system. A qualitative study conducted on 22 South Asian adolescent girls in Canada showed that their parents and communities have more stringent rules for female socialization than any other community in the country (Talbani & Hasanali, 2000).\n\nInterpersonal and socio-cultural norms are the ultimate determinants of the nature of GBV. For instance, in typical South Asian culture, male figures are traditionally seen as an embodiment of inheritance, entitlement, and power where domestic violence (in some form or other) is justified (to a great extent) by long-standing cultural and societal values and is seen as an inevitable part of conjugal life resulting from time to time as a natural process in a rather happily living couple. Thus, the depth of the problem is subject to being over- and/or underestimated or completely neglected depending on the depth of individual’s perception of and willingness to acknowledging it as a problem. According to a study conducted on a slum population in Mumbai, the 35% of women indicated that wife-beating is justifiable if they disrespected their in-laws or argued with their husband, or failed to provide good food, housework and childcare, or went out without permission (Das et al., 2013). It is not unusual for South Asians women to prefer suffering spousal violence than going to court fearing that this may incur in social stigma, which is more excruciating and would worsen their family problem. Researchers noted that South Asian women are exceptionally inclined to maintaining cultural values of family structure, marriage, and religious practice even when trying to adjust to different gender role expectations, language, and cultural values in a foreign country (Inman et al., 2001). A similar tendency is observed among immigrant South Asian women of silently accepting abusive behavior inside family and avoiding to seek consultation and legal services.\n\n\nNature, prevalence and risk factors of GBV in South Asia\n\nIn general terms, GBV encompasses following categories: IPV (denotes relationship to perpetrator), domestic violence (denotes location of the abuse) and violence against women (denotes the sex of the survivor) (WHO, 2005), each of which can take various forms of physical and mental torture e.g. physical manhandling, financial threats, sexual abuse, emotional blackmailing. The World Health Organization (WHO) defines violence as “the result of the complex interplay of individual, relationship, social, cultural and environmental factors” (Krug et al., 2002). The categorization of GBV provided by Article-2 of (Declaration on the Elimination of Violence against Women ) (United Nations, 1993) is as follows:\n\nViolence against women shall be understood to encompass, but not be limited to, the following:\n\n1. Physical, sexual and psychological violence occurring in the family, including battering, sexual abuse of female children in the household, dowry-related violence, marital rape, female genital mutilation and other traditional practices harmful to women, non-spousal violence and violence related to exploitation.\n\n2. Physical, sexual and psychological violence occurring within the general community, including rape, sexual abuse, sexual harassment and intimidation at work, in educational institutions and elsewhere, trafficking in women and forced prostitution.\n\n3. Physical, sexual and psychological violence perpetrated or condoned by the State, wherever it occurs.\n\nObtaining accurate data on GBV has several inherent challenges that lie in the variations in definitions and methodology and especially in underreporting by the victims as the topic is usually associated with element of shame and embarrassment. In a population-based comparative study conducted in India, men were found to report higher prevalence of all forms of violence compared to women (Babu & Kar, 2009). According to the WHO multi-country study, women in Japan were the least likely to suffer domestic violence while the greatest prevalence of violence was reported in Bangladesh, Ethiopia, Peru and the United Republic of Tanzania (WHO, 2005).\n\nApart from violence incidences in domestic setting, girls and women trafficking is another serious gender and human right issue which is highly prevalent across South Asia. Shockingly, families and friends/acquaintances constitute the majority of perpetrators behind women trafficking, with Nepal claimed to face one of the worst trafficking scenario in the world (Silverman et al., 2007). According to a study based in Nepal, in about 80% of the cases, the perpetrator used to be a member of the victim’s own family (Dhakal, 2008). Women and girls (South Asian) rescued from brothels in Mumbai reported that over half of the victims were trafficked by individuals previously known to them (Silverman et al., 2007). Study conducted on a South Asian brothel population found that one fourth of the subjects were trafficked through someone they knew and suffered more frequent violent incidents (Sarkar, 2008).\n\nPrevalence. Violence against women, in any form, is highly prevalent across South Asia (Table 1) (Hadi, 2000 and Naved & Akhtar, 2008). In Northern India, 25% of husbands reported having perpetrated physical violence against their wives during the preceding year and 30% reported having committed sexual violence (Ahmad et al., 2015). In a Bangladesh study, more than one in three men reported physical violence, sexual violence, or both against their wives in the past 12 months (Fulu et al., 2013), one of the worst result for any South Asian Nation. According to the Violence against Women Survey in Bangladesh (VAW 2011), the first nationwide survey in the country conducted by the Bangladesh Bureau of Statistics (BBS) in collaboration with the United Nations Population Fund (UNDP), nearly 77% of the study subjects were abused in last 12 months and only 8% of women said they had never been abused by a man other than their husbands. In Pakistan, the Human Rights Commission (HRCP) estimated the prevalence of domestic violence as 65%, one-third (30.4%) of which was reported to be sexual violence (HRCP, 2004). Honor killing (HK) is another serious public health concern in the country with an estimated 1957 HK events taking place from 2004 to 2007, according to a study based on newspaper reports (Nasrullah et al., 2009). Sri Lanka, despite having the highest literacy rate (both sexes) and the best gender parity situation (Global gender index 2014) in South Asia, the prevalence of IPV remains shockingly high (40%, as of 2010) (Jayatilleke et al., 2010). In Nepal, one-third (35%) of all married women are reported to experience domestic violence.\n\nSource: UN Women. Violence against Women Prevalence Data: Surveys by Country (As of December 2012). DHS: Demographic and Health Survey\n\n\nRisk factors of domestic violence in South Asia\n\nThe causes of GBV are social, economic, cultural, political and religious (Sanjel, 2013). According to locally available reports (reports not circulated internationally), the causes of domestic violence vary greatly from simple matters as failing to finish cooking on time to more important matters such as inability to produce male offspring or infertility. Reviews on GBV in South Asian countries summarized that GBV results most commonly from illiteracy to low education; economic, educational and cultural inferiority of women, family size, number of male children, women’s acceptance of violence, and marital discord (Babu & Kar, 2009). Women belonging to lower castes, who are illiterate, and come from poor economic backgrounds are also more at risk of experiencing violence than others (Babu & Kar, 2009). India alone saw almost 7,000 dowry related deaths in 2005 (Garcia-Moreno, 2009). In Nepal, among the various causes of domestic violence, dowry-related hostilities, second marriages by husbands, assaults on women accused of being witches, and disputes involving properties are the main reasons (Gao et al., 2012, and Dhakal, 2008). A study in Bangladesh found that young age (20–29 years) illiteracy and poverty increased a married women's risk of being sexually abused and the risk is less among women who participate in credit programs and financially contribute to their families (Hadi, 2000). Educated and working women suffer less violence due to their greater economic independence and better understanding of rights and higher access to resources. A study conducted in Urban Slums in Lahore, showed that family affairs particularly issues with in-laws, poor house management, lack of proper care of children, bringing insufficient dowry, financial problems, an act against the will of husband, and inability to give birth to a male child were associated to domestic violence against women (Nasrullah et al., 2009).\n\n\nGBV and women’s health in South Asia\n\nGBV is a major threat to global public health and poses significant barriers to the advancement to women’s health. GBV can cause a host of acute to long-term health effects with serious and aggravating consequences on reproductive, sexual and psychological status: physical injuries and disabilities, spread of sexually transmitted diseases (STDs), unintended pregnancies, gynecological problems, mild to severe mood disorder and suicide (Kaur & Garg, 2008). In a study encompassing all the six zones of India, 37% of the women reported a high prevalence of psychological violence (Kaur & Garg, 2008).\n\nGlobally, GBV places women in significantly susceptible to human immunodeficiency virus (HIV) infection. In sub-Saharan Africa for instance women aged 15–24 are about eight times more likely to be HIV positive compared to their male counterparts. In Asia, women account for a growing proportion of HIV infections: from 21% in 1990 to 35% in 2009 (UNAIDS, 2010). Though the rate of HIV in South Asia is comparatively lower than in other developing countries, future risk of spread remains high given the rising incidence of GBV in this region. Researchers at Harvard University found that in India the risk of HIV infection among married women who experienced domestic violence were about four times higher in comparison to those who did not face any violence (Silverman et al., 2008).\n\nIn recent years there has been a rising interest in women's reproductive health development related researches and programs to enhance reproductive health across the globe (Kaddour et al., 2005). Universal access to quality sexual and reproductive health (SRH) services is regarded as a key component to fulfilling many (if not all) of the MDGs (Millennium Development Goals), especially the ones relating to maternal and child health, HIV and gender equity. Researches demonstrated that SRH is of fundamental importance to national health and to the economic development at large (Mishra & Lohiya, 2016). Despite the governments solid endeavor to meeting the health related MDGs, those concerning reproductive health remain largely unmet.\n\nLiterature review on reproductive health indicates an escalating concern on domestic violence and its repercussion on health and overall being of women and children. Researchers across various global regions have identified GBV as a major contributor to poor reproductive outcomes for women, with abused women twice as likely to report unintended pregnancy and three times more likely to give birth as an adolescent compared to those not experiencing any violence (Silverman & Raj, 2014). A similar scenario exists across South Asia where GBV constitutes a significant share of all SRH related burden. A study based on NFHS in India (National Family Health Survey- 1998–99) revealed that women who were physically mistreated by their husbands were almost twice as likely to experience unintended pregnancies (Begum et al., 2010). Another NFHS (2005–06) based study including 65610 married women in reproductive age (15–49 years) reported that 23.9% of women experienced at least one form of IPV and had pregnancy related complicacies (Winter & Stephenson, 2013). Apart from direct impact on health status, GBV tends to undermine women’s reproductive control, inadequate communication and cooperation regarding family planning, sexual and reproductive health issues, poor delivery preparedness and pre- and postnatal care. Almost all IPV perpetration in South Asia occurs within marriage (Fulu et al., 2013). Results from a cross-sectional study conducted in Lahore (Pakistan) showed that women who experienced domestic violence were more likely to suffer from poor prenatal care, unplanned pregnancies and poor self-reported reproductive health and lack of cooperation in contraceptive use from husbands as compared with non-abused women (Zakar et al., 2012). A growing evidence supports the fact that state of maternity does not have any significant protective effect on women from violent spousal behavior. The incidence of domestic violence during maternity in India was reported to be 21–28% (Das et al., 2013). A more recent evidence from Uttar Pradesh shows that ~ 47% of the women had experienced some form of violence during their last pregnancy (Ahmad et al., 2015). Obstetricians in Pakistan reported that more than 30% of Pakistani women suffer from some form of domestic violence. This situation is especially vulnerable for women who become mother at a premature age. The average median age at first birth in South Asia is far lower than observed in other parts of Asia (17.9 in Bangladesh, 19.9 years in Nepal, 21.8 years in Pakistan) (NFHS-3, 2005–06). Moreover, premature marriage and childbearing cut short young women's educational and employment opportunities and increase the likelihood of lifelong subordination to their husbands.\n\nIn addition to GBV, more abhorrent forms of violence are female feticide and sex-selective abortion, which are becoming increasingly popular in South Asia and represent emerging challenges for achieving gender equity and enhancing women’s health and human rights. In addition to compromising the efforts to promote women’s health, feticide itself is a precarious process and increases the risk of maternal morbidity and mortality. South Asian societies exhibit an explicit leaning for male children usually to secure family wealth, preserving family line and avoiding the expenses of dowry. Gender imbalance (skewed sex ratio) has already began to take shape in India (Notably in Punjab, Rajasthan and Haryana) and to cause adverse social impacts in terms of age, caste, religion. Young male citizens are predicted to exceed their female counter parts by a staggering 10–20% by next 20 years.\n\n\nRole of healthcare system in tackling GBV\n\nThe World Health Assembly recognizes GBV as a major public health issue that warrants immediate attention from governments and healthcare organizations. A literature review study by USAID suggested that GBV has implications for almost every aspects of health policy and programming ranging from primary care to reproductive health programs (Guedes, 2004). Studies have also shown that gender-sensitive healthcare policies can make a difference in the lives of those who experience abuse (Garg & Singh, 2013). Though the problem is essentially social, the healthcare system holds a crucial role in the prevention and intervention of GBV starting from ensuring effective screening of victims, informing people about the health impacts to meeting their physical and psychological health needs. Compared to communicating with surveyors and administration, people naturally have more confidence and are less introvert and withdrawn with caregivers because of the characteristic psychological support and reliance that people tend to attach on caregivers which can be leveraged to improve the screening, treatment, rehabilitation procedure and disseminating violence related advices. Incorporating social media tools in public health service programs could serve an equally vital part in abating the complexities of stigma and culture of silence associated with GBV which is also fundamental to promote care-seeking behavior. Interdisciplinary team health specialists including PHWs (Public health workers), clinicians and medical anthropologists can mutually cooperate in classifying and categorizing the health consequences of GBV understanding the urgency of the situation, making critical referrals accordingly and designing easily accessible and effective caregiving models especially for those living in remote areas.\n\nEnabling healthcare to GBV prevention however doesn’t guarantee the end of abusive behavior. A qualitative study conducted in Brazil showed that the physicians were unable to handle the situation as patients suffered repeated torturing with worsening health conditions despite regular treatment, and complained about society’s indifference regarding the matter (Elisabeth et al., 2013). To break recurring abuse among the victims, community clinics working in collaboration with NGOs offer the potential to reduce the physical and communication distance between victims and the legal service systems, NGOs might be better equipped to interact with the administrative bodies. On the policy front, the addition of a gender dimension in a broader public health policy framework and strategic resource allocation to promote gender-sensitive social and healthcare policies should target the advancement of women’s psychological and reproductive health.\n\n\nConclusion and policy recommendations\n\nThis review provides an update on GBV in south Asia and reveals that GBV in all its forms and the contextual determinants are highly prevalent across this region. GBV is a ubiquitous public health issue around the globe, however bears special significance in the context of South Asia owing to its deep-rooted socio-cultural barriers compounded by poor healthcare and social infrastructure and lack of institutional accountability. GBV, as a multidimensional problem, calls for a multidimensional solution. The study also highlights that GBV preventive measure must focus on strategic areas of women empowerment such as leveraging gender and sex education, job creation, designing gender-sensitive public policy, law enforcement and strengthening administrative transparency. By drawing evidence from locally available resources and published research articles, this study concludes by making the following policy recommendations that:\n\n1. A growing evidence suggests that poverty and lack of access to productive resources among women significantly increase the risk of domestic violence (DV) and sex trafficking (Babu & Kar, 2009; Fulu et al., 2013). Microcredit programs positively correlate with decreased violent events (Hadi, 2000) and minimize gender disparity in funding. Studies have shown that in South Africa, Intervention with Microfinance for AIDS and Gender Equity (IMAGE) has reduced the incidence of IPV by half (Kim et al., 2007). As the birthplace of microfinance, gender based microfinance programs can do wonders for south Asian women in helping to improve economic autonomy, decision-making power, right to health, freedom from discrimination. However, special monitoring systems must be in place to ensure that women have full control over the economic opportunity they are receiving, have equal access to inputs and face no wage discrimination.\n\n2. Evidently, people (most) are not aware of the various health and socio-economic impacts of GBV and its negative repercussions on children, other family members and overall wellbeing of the family. Nationwide anti-violence campaign and behavior change intervention programs should target awareness creation to reduce gender biases and encourage gender-sensitive behavior, eradication of health and gender-related illiteracy and ensure men’s participation at levels of such programs.\n\n3. The impact of skewed sex ratio is already noticeable in term of demographic imbalance and has the potential to cause huge social externalities if unchecked for long. In some areas, the sex ratio of females to males has dropped to less than 8:1 (Ahmad, 2010). Controlling prenatal sex screening and sex-selective abortion technologies therefore presents an urgent imperative. Female feticide embodies a flagrant violation of human rights and decline of moral values and ethics on which rests the very foundation of civilization. This odious act must be prevented with urgency through introducing rigorous policy instruments.\n\n4. Frail law enforcement and prosecution systems is a significant challenge to confronting GBV. According a nine-country study in the Asia Pacific region (Including Bangladesh and Sri Lanka) conducted jointly by UNDP, UNFPA (United Nations Population Fund), UN Women and UN in 2013, 72–97% of men who had committed rape were never punished which confirms a serious unaccountability of the human rights law. To overcome the lack of policy and managerial transparencies, it is imperative to establish a robust domestic legal system and independent human right institution at local and national level.\n\n5. Globally, resources allocated by the government to health-promoting activities are very limited compared to investments in medical care (Piroska et al., 2006). The operational financing and the integration of healthcare system with GBV agenda depend greatly on political commitment for adequate resource allocation, especially when developing a new branch in public health with sufficient human resources trained in understanding the context, meeting the unique needs of GBV patients and building physical infrastructures such as transportation, community service centers, special examination and counseling rooms. The healthcare expenditure among abused women is significantly higher compared to non-abused women. Special attention must be paid so that financial barrier doesn’t compromise the healthcare service of the marginalized population.\n\n6. Progress can be thwarted by inadequate information and underreporting the prevalence of GBV. Conducting national survey on regular basis by developing an effective screening strategy to identify GBV cases is crucial to making culturally tailored GBV prevention and intervention strategies which will assist greatly in assessing and monitoring progress towards gender equity.\n\n7. Long-term success against GBV will require the adoption an interdisciplinary operational framework by incorporating a wide spectrum of crosscutting strategies and enhancing multi-stakeholder engagement in the overall development process. Political commitment are vital to minimize policy related barriers and developing a gender-friendly political environment. Creating a greater synergy between government and civil society organizations (CSOs) is equally essential to understanding the barriers to implementation of policies and how they can be overcome.\n\n\nStrengths and limitations of the study\n\nThis study has drawn evidence from locally available gray literature which portray the facts better but are not usually considered for academic researches. Current state of GBV in five South Asian countries were reviewed: Bangladesh, India, Nepal, Pakistan, and Sri Lanka. Clinical studies and any studies before 2000 were not included. The most recent studies and studies by native authors were given priority. Only population-based studies conducted on native South Asian women were considered for this review, studies on migrant South Asians were not included. Health effects GBV was limited to reproductive health.",
"appendix": "Author contributions\n\n\n\nGB conceptualized the study. GB and SS were responsible for literature search and selection of suitable research materials. All authors contributed to drafting the manuscript. GB and SY contributed to critical reviewing and modifications. All authors read the final version of the manuscript and approved it 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\nAcknowledgements\n\nSincere thanks to many of our colleagues from all the countries included in the study who helped by providing useful study materials and logistical support pertinent to this study.\n\n\nReferences\n\nAhmad J, Khan ME, Mozumdar A, et al.: Gender-Based Violence in Rural Uttar Pradesh, India: Prevalence and Association With Reproductive Health Behaviors. J Interpers Violence. 2015; pii: 0886260515584341. PubMed Abstract | Publisher Full Text\n\nAhmad J, Khan ME, Mozumdar A, et al.: Gender-Based Violence in Rural Uttar Pradesh, India: Prevalence and Association With Reproductive Health Behaviors. J Interpers Violence. 2015; pii: 0886260515584341. PubMed Abstract | Publisher Full Text\n\nAhmad N: Female feticide in India. Issues Law Med. 2010; 26(1): 13–29. PubMed Abstract\n\nBabu BV, Kar SK: Domestic violence against women in eastern India: a population-based study on prevalence and related issues. BMC Public Health. 2009; 9: 129. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBegum S, Dwivedi SN, Pandey A, et al.: Association between domestic violence and unintended pregnancies in India: findings from the National Family Health Survey-2 data. Natl Med J India. 2010; 23(4): 198–200. PubMed Abstract\n\nDas S, Bapat U, Shah More N, et al.: Intimate partner violence against women during and after pregnancy : a cross-sectional study in Mumbai slums. BMC Public Health. 2013; 13: 817. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeclaration on the Elimination of Violence against Women. United Nations General Assembly. A/RES/48/104. 1993. Reference Source\n\nDhakal S: Nepalese women under the shadow of domestic violence. Lancet. 2008; 371(9612): 547–548. PubMed Abstract | Publisher Full Text\n\nFulu E, Jewkes R, Roselli T, et al.: Prevalence of and factors associated with male perpetration of intimate partner violence: findings from the UN Multi-country Cross-sectional Study on Men and Violence in Asia and the Pacific. Lancet Glob Health. 2013; 1(4): e187–e207. PubMed Abstract | Publisher Full Text\n\nGao E, Zuo X, Wang L, et al.: How does traditional Confucian culture influence adolescents' sexual behavior in three Asian cities? J Adolesc Health. 2012; 50(3 Suppl): S12–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarcia-Moreno C: Gender inequality and fire-related deaths in India. Lancet. 2009; 373(9671): 1230–1231. PubMed Abstract | Publisher Full Text\n\nGarg S, Singh R: Gender-violence and health care: How health system can step in. Indian J Public Health. 2013; 57(1): 4–7. PubMed Abstract | Publisher Full Text\n\nGuedes A: Addressing Gender-Based Violence from the Reproductive Health/HIV Sector: A Literature Review and Analysis. Washington, DC: USAID, Bureau for Global Health. 2004. Reference Source\n\nHadi A: Prevalence and correlates of the risk of marital sexual violence in Bangladesh. J Interpers Violence. 2000; 15(8): 787–805. Publisher Full Text\n\nHuman rights commission of Pakistan: Annual report. Violence against women in Pakistan. 2004.\n\nIIPS, International Institute for Population Sciences (IIPS) and Macro International: National Family Health Survey (NFHS-3), 2005–06. Mumbai, India: 2007; 1. Reference Source\n\nInman AG, Ladany N, Constantine MG, et al.: Development and preliminary validation of the cultural values conflict scale for South Asian women. J Couns Psychol. 2001; 48(1): 17–27. Publisher Full Text\n\nJayatilleke AC, Poudel KC, Yasuoka J, et al.: Intimate partner violence in Sri Lanka. Biosci Trends. 2010; 4(3): 90–5. PubMed Abstract\n\nJohnston HB, Naved RT: Spousal violence in Bangladesh: a call for a public-health response. J Health Popul Nutr. 2008; 26(3): 366–377. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaddour A, Hafez R, Zurayk H: Women's perceptions of reproductive health in three communities around Beirut, Lebanon. Reprod Health Matters. 2005; 13(25): 34–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKamimura A, Parekh A, Olson LM: Health indicators, social support, and intimate partner violence among women utilizing services at a community organization. Womens Health Issues. 2013; 23: 3: e179–e185. PubMed Abstract | Publisher Full Text\n\nKaur R, Garg S: Addressing domestic violence against women: an unfinished agenda. Indian J Community Med. 2008; 33(2): 73–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim JC, Watts CH, Hargreaves JR, et al.: Understanding the impact of a microfinance-based intervention on women’s empowerment and the reduction of intimate partner violence in South Africa. Am J Public Health. 2007; 97(10): 1794–1802. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKrug GE, Dahlberg LL, James Mercy A, et al.: World Report on Violence and Health. Geneva: World Health Organization (WHO). 2002. Reference Source\n\nMishra PK, Lohiya NK: Prioritizing reproductive health: Can it be the real game changer for India? J Reprod Health Med. 2016; 2(2): 1–3. (In press). Publisher Full Text\n\nMurthy P: Women's Global Health and Human Rights Couverture ISBN. 354, ISBN-13: 978-0763756314. Reference Source\n\nNasrullah M, Haqqi S, Cummings KJ: The epidemiological patterns of honour killing of women in Pakistan. Eur J Public Health. 2009; 19(2): 193–7. PubMed Abstract | Publisher Full Text\n\nNaved RT, Akhtar N: Spousal violence against women and suicidal ideation in Bangladesh. Womens Health Issues. 2008; 18(6): 442–52. PubMed Abstract | Publisher Full Text\n\nNiaz U: Violence against women in South Asian countries. Arch Womens Ment Health. 2003; 6(3): 173–84. PubMed Abstract | Publisher Full Text\n\nPrevention of Domestic Violence Act (PDVA, 2005). (Accessed on 17/-4/2016). Reference Source\n\nSanjel S: Gender-based violence: a crucial challenge for public health. Kathmandu Univ Med J (KUMJ). 2013; 11(42): 179–84. PubMed Abstract | Publisher Full Text\n\nSarkar NN: The impact of intimate partner violence on women's reproductive health and pregnancy outcome. J Obstet Gynaecol. 2008; 28(3): 266–71. PubMed Abstract | Publisher Full Text\n\nSilverman JG, Decker MR, Gupta J, et al.: Experiences of sex trafficking victims in Mumbai, India. Int J Gynaecol Obstet. 2007; 97(3): 221–6. PubMed Abstract | Publisher Full Text\n\nSilverman JG, Decker MR, Saggurti N, et al.: Intimate partner violence and HIV infection among married Indian women. JAMA. 2008; 300(6): 703–10. PubMed Abstract | Publisher Full Text\n\nSilverman JG, Raj A: Intimate partner violence and reproductive coercion: global barriers to women's reproductive control. PLoS Med. 2014; 11(9): e1001723. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSolotaroff JL, Prabha RP: Violence against Women and Girls: Lessons from South Asia. World Bank Group, Washington. 2014. Reference Source\n\nSudha S, Morrison S: Marital violence and women's reproductive health care in Uttar Pradesh, India. Womens Health Issues. 2011; 21(3): 214–21. PubMed Abstract | Publisher Full Text\n\nTalbani A, Hasanali P: Adolescent females between tradition and modernity: gender role socialization in South Asian immigrant culture. J Adolesc. 2000; 23(5): 615–27. PubMed Abstract | Publisher Full Text\n\nWinter A, Stephenson R: Intimate partner violence and symptoms of reproductive tract infections among married Indian women. Int J Gynaecol Obstet. 2013; 121(3): 218–223. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization: WHO Multi-country Study on Women's Health and Domestic Violence Against Women. Geneva: WHO; 2005. Reference Source\n\nZakar R, Zakar MZ, Mikolajczyk R, et al.: Intimate partner violence and its association with women's reproductive health in Pakistan. Int J Gynaecol Obstet. 2012; 117(1): 10–14. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13715",
"date": "12 Oct 2016",
"name": "Shakuntala Amirchand Chhabra",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is on an important subject for reviews and studies, however it needs major revision.\nAs one looks at the title and tries to understand what the authors wish to share, the expectations given by the title are different than what the article provides. The review methodology mentions the Cochrane Database of Systematic Reviews – the expectation was that they would be ordered in relation to title, socio-cultural aspects of gender based violence and relevant information, the introduction of the subject as per the title, the frequency of GBV, which socio-cultural aspects affect GBV in the proper order, what are the effects on women’s health, and to what extent.\n\nHowever, as one goes through the article (if one overlooks issues related to grammar, language, and the repetition of sentences) it seems a lot was needed, and that the study format as it was designed executed and discussed is different to the design as per the title. The current structure of Introduction, Incidence, Risk Factors, Attitude aspects (with studies based on interviews, about GBV, visible, invisible), the Impact of GBV on non-pregnant and pregnant women’s health, means that some paragraphs are not really relevant to the title.\nIn addition, there are also references included in the Conclusion section.\nOverall, in my opinion both the design and execution could have been much better, and the discussion should be more orderly also. The grammar and language should have been looked over, for example avoiding repeated sentences.",
"responses": []
}
] | 1
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https://f1000research.com/articles/5-802
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https://f1000research.com/articles/4-231/v1
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13 Jul 15
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{
"type": "Review",
"title": "Enteropathogenic E. coli: breaking the intestinal tight junction barrier",
"authors": [
"Anand Prakash Singh",
"Saima Aijaz",
"Anand Prakash Singh"
],
"abstract": "Enteropathogenic E. coli (EPEC) causes acute intestinal infections in infants in the developing world. Infection typically spreads through contaminated food and water and leads to severe, watery diarrhea. EPEC attaches to the intestinal epithelial cells and directly injects virulence factors which modulate multiple signaling pathways leading to host cell dysfunction. However, the molecular mechanisms that regulate the onset of diarrhea are poorly defined. A major target of EPEC is the host cell tight junction complex which acts as a barrier and regulates the passage of water and solutes through the paracellular space. In this review, we focus on the EPEC effectors that target the epithelial barrier, alter its functions and contribute to leakage through the tight junctions.",
"keywords": [
"Enteropathogenic E. coli",
"tight junctions",
"paracellular permeability."
],
"content": "Introduction\n\nEPEC causes diarrhea in infants in developing countries and is responsible for more than a million deaths annually1,2. EPEC infection results in excessive loss of water and electrolytes from the body leading to dehydration and death3. However, the underlying molecular mechanisms are not completely understood. In intestinal epithelial cells, tight junctions regulate the passage of water and solutes and are targeted by EPEC4. EPEC directly injects virulence factors into the host cells which target multiple signaling pathways and some have been linked to tight junction disruption5. In this review, we focus only on the EPEC effectors reported to be involved in the disruption of tight junctions.\n\n\nDiscussion\n\nEPEC attaches to the apical surface of the intestinal epithelial cells and effaces the microvilli causing localized lesions6,7. EPEC uses a type three secretion system to inject at least twenty five effector proteins into the host cells8. Many of the effectors are encoded by genes located on a pathogenicity island called the locus of enterocyte effacement (LEE) although non-LEE encoded effectors have also been identified9. One of the effectors, Tir (Translocated intimin receptor) is inserted into the host plasma membrane where it serves as a receptor for intimin, the outer membrane protein of EPEC10. The LEE- and non-LEE- encoded effectors disrupt host cell function by cytoskeletal reorganization, mitochondrial dysfunction, protein transport defects, suppression of immune responses and epithelial barrier disruption8. Since the intestinal tight junctions regulate paracellular permeability, EPEC-mediated disruption of this complex likely contributes to the onset of diarrhea.\n\nTight junctions seal adjacent epithelial cells and act as (i) a gate that selectively regulates the passage of ions and solutes through the paracellular space and (ii) a fence that prevents the intermixing of apical and basal plasma membrane proteins thereby maintaining cell polarity11,12. The tight junction complex consists of transmembrane proteins, adaptor proteins, small GTPases, kinases, phosphatases, transcriptional and post-transcriptional regulators11,12. The transmembrane proteins regulate cell-cell adhesion and are linked to the cytoskeleton through the adaptor proteins11,12. Paracellular permeability is regulated by the transmembrane proteins belonging to the Marvel-domain containing protein family (occludin, Tricellulin/MarvelD2 and MarvelD3) as well as by members of the claudin family11,13. Additionally, permeability is regulated by tight junction-associated guanine-nucleotide exchange factors for Rho GTPases through the modulation of the actin cytoskeleton11–13.\n\nLeakage through tight junctions occurs in diarrhea and the EPEC effectors EspF, Map, EspG/G2 and NleA have been implicated in the disruption of host cell tight junctions14–16.\n\nEspF is a multifunctional effector which disrupts the tight junction barrier14. The N-terminus of EspF contains mitochondrial and nucleolus targeting sequences which direct EspF to the mitochondria and nucleolus respectively where it alters their functions17. The C-terminus of EspF contains three proline rich repeats with binding sites for eukaryotic sorting nexin 9 (SNX9) and neuronal Wiskott-Aldrich syndrome protein (N-WASP)17. Binding of EspF with SNX9 is required for its recruitment to the plasma membrane but is not sufficient for tight junction disruption as EspF mutants deficient in SNX9 binding also disrupt tight junctions18. Binding of EspF with N-WASP triggers the activation of the Arp2/3 complex leading to actin polymerization18. EspF inactivates the sodium-D-glucose co-transporter SGLT-1 through co-operative actions of other EPEC effectors Map, Tir and intimin and also inhibits the activity of the Na+/H+ exchanger NHE317,19. EspF from the rabbit EPEC strain E22 has been shown to bind actin and recruit the tight junction proteins ZO-1 and ZO-2 into actin pedestals20 while EspF from the mouse EPEC strain C. rodentium is involved in the internalization of claudin-1, -3 and -5 in vivo21 causing tight junction disruption.\n\nThe EPEC effector Map cooperates with EspF in the disruption of tight junctions14. Like EspF, Map is also targeted to the mitochondria where it alters mitochondrial functions14. Map activates the Cdc42 GTPase by functioning as its GEF (guanine-nucleotide exchange factor) leading to the formation of transient filopodia22. The C-terminus of Map contains a TRL (Thr-Arg-Leu) motif through which it interacts with EBP50 (ERM-binding phosphoprotein 50) also called Na+/H+ exchanger regulatory factor 1 (NHERF1)23. Binding with EBP50 recruits the actin scaffold protein ezrin to this complex linking Map to the actin cytoskeleton. Map also interacts with Na+/H+ exchanger regulatory factor 2 (NHERF2) and in association with EspF inactivates SGLT-119,24. The precise mechanism through which Map disrupts tight junctions is not known but it likely occurs through modulation of the actin cytoskeleton.\n\nAnother EPEC effector that binds with NHERF2 is NleA24. NleA has been reported to increase paracellular permeability by disrupting the tight junction proteins ZO-1 and occludin16. NleA inhibits protein secretion from the ER (endoplasmic reticulum) to the Golgi by direct interaction with Sec24, a subunit of the coat protein complex II (COP-II)25. It has been proposed that NleA inhibits the transport of newly synthesized tight junction proteins disrupting the barrier.\n\nThe EPEC effector EspG and its homolog EspG2 have been shown to increase the permeability of small tracers through the tight junctions15. EspG binds the Golgi matrix protein GM130 disrupting the Golgi structure26. Additionally, EspG deregulates the small GTPases Arf1/6 (ADP ribosylation factor 1/6) and Rab1 causing arrest of protein trafficking from the ER to the Golgi27,28. EspG also binds p21-activated kinase at the same site required for Rab1 binding28,29 linking the microtubules to this signaling axis indicating that EspG selectively regulates multiple signaling pathways ultimately disrupting tight junctions.\n\n\nConclusions\n\nCoordinated actions of the EPEC effectors EspF, Map, NleA and EspG disrupt tight junctions. With the exception of EspF, which forms a complex with ZO-1/ZO-2, none of the other effectors have been reported to interact with any tight junction protein. Therefore, their role in the disruption of tight junctions and onset of diarrhea is possibly due to (i) modulation of the actin cytoskeleton (EspF, Map) or the microtubules (EspG); (ii) inhibition of protein trafficking from the ER to the Golgi (EspG, NleA); (iii) interference with the functions of the Na+/H+ exchanger (EspF), SGLT-1 (EspF, Map), NHERF-1, -2 (Map, NleA) and (iv) deregulation of aquaporins (EspF, EspG)30.",
"appendix": "Author contributions\n\n\n\nBoth authors contributed in the writing of this manuscript and approved the final version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nLanata CF, Fischer-Walker CL, Olascoaga AC, et al.: Global causes of diarrheal disease mortality in children <5 years of age: a systematic review. PLoS One. 2013; 8(9): e72788. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOchoa TJ, Contreras CA: Enteropathogenic Escherichia coli infection in children. Curr Opin Infect Dis. 2011; 24(5): 478–483. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNataro JP, Kaper JB: Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998; 11(1): 142–201. PubMed Abstract | Free Full Text\n\nGuttman JA, Finlay BB: Subcellular alterations that lead to diarrhea during bacterial pathogenesis. Trends Microbiol. 2008; 16(11): 535–542. PubMed Abstract | Publisher Full Text\n\nDean P, Kenny B: The effector repertoire of enteropathogenic E. coli: ganging up on the host cell. Curr Opin Microbiol. 2009; 12(1): 101–109. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVallance BA, Finlay BB: Exploitation of host cells by enteropathogenic Escherichia coli. Proc Natl Acad Sci U S A. 2000; 97(16): 8799–8806. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWong ARC, Pearson JS, Bright MD, et al.: Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements. Mol Microbiol. 2011; 80(6): 1420–1438. PubMed Abstract | Publisher Full Text\n\nDean P, Maresca M, Kenny B: EPEC’s weapons of mass subversion. Curr Opin Microbiol. 2005; 8(1): 28–34. PubMed Abstract | Publisher Full Text\n\nIguchi A, Thomson NR, Ogura Y, et al.: Complete genome sequence and comparative genome analysis of enteropathogenic Escherichia coli O127:H6 strain E2348/69. J Bacteriol. 2009; 191(1): 347–354. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrankel G, Phillips AD: Attaching effacing Escherichia coli and paradigms of Tir-triggered actin polymerization: getting off the pedestal. Cell Microbiol. 2008; 10(3): 549–556. PubMed Abstract | Publisher Full Text\n\nAijaz S, Balda MS, Matter K: Tight junctions: molecular architecture and function. Int Rev Cytol. 2006; 248: 261–298. PubMed Abstract | Publisher Full Text\n\nVan Itallie CM, Anderson JM: Architecture of tight junctions and principles of molecular composition. Semin Cell Dev Biol. 2014; 36: 157–165. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKrug SM, Schulzke JD, Fromm M: Tight junction, selective permeability, and related diseases. Semin Cell Dev Biol. 2014; 36: 166–176. PubMed Abstract | Publisher Full Text\n\nDean P, Kenny B: Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein. Mol Microbiol. 2004; 54(3): 665–675. PubMed Abstract | Publisher Full Text\n\nMatsuzawa T, Kuwae A, Abe A: Enteropathogenic Escherichia coli type III effectors EspG and EspG2 alter epithelial paracellular permeability. Infect Immun. 2005; 73(10): 6283–6289. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThanabalasuriar A, Koutsouris A, Weflen A, et al.: The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic Escherichia coli. Cell Microbiol. 2010; 12(1): 31–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHolmes A, Mühlen S, Roe AJ, et al.: The EspF effector, a bacterial pathogen’s Swiss army knife. Infect Immun. 2010; 78(11): 4445–4453. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlto NM, Weflen AW, Rardin MJ, et al.: The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways. J Cell Biol. 2007; 178(7): 1265–1278. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDean P, Maresca M, Schüller S, et al.: Potent diarrheagenic mechanism mediated by the cooperative action of three enteropathogenic Escherichia coli-injected effector proteins. Proc Natl Acad Sci U S A. 2006; 103(6): 1876–1881. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeralta-Ramirez J, Hernandez JM, Manning-Cela R, et al.: EspF Interacts with nucleation-promoting factors to recruit junctional proteins into pedestals for pedestal maturation and disruption of paracellular permeability. Infect Immun. 2008; 76(9): 3854–3868. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuttman JA, Li Y, Wickham ME, et al.: Attaching and effacing pathogen-induced tight junction disruption in vivo. Cell Microbiol. 2006; 8(4): 634–645. PubMed Abstract | Publisher Full Text\n\nHuang Z, Sutton SE, Wallenfang, AJ, et al.: Structural insights into host GTPase isoform selection by a family of bacterial GEF mimics. Nat Struct Mol Biol. 2009; 16(8): 853–860. PubMed Abstract | Publisher Full Text\n\nSimpson N, Shaw R, Crepin VF, et al.: The Enteropathogenic Escherichia coli type III secretion system effector Map binds EBP50/NHERF1: implication for cell signalling and diarrhoea. Mol Microbiol. 2006; 60(2): 349–363. PubMed Abstract | Publisher Full Text\n\nMartinez E, Schroeder GN, Berger CN, et al.: Binding to Na+/H+ exchanger regulatory factor 2 (NHERF2) affects trafficking and function of the enteropathogenic Escherichia coli type III secretion system effectors Map, EspI and NleH. Cell Microbiol. 2010; 12(12): 1718–1731. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim J, Thanabalasuriar A, Chaworth-Musters T, et al.: The bacterial virulence factor NleA inhibits cellular protein secretion by disrupting mammalian COPII function. Cell Host Microbe. 2007; 2(3): 160–171. PubMed Abstract | Publisher Full Text\n\nClements A, Smollett K, Lee SF, et al.: EspG of enteropathogenic and enterohemorrhagic E. coli binds the Golgi matrix protein GM130 and disrupts the Golgi structure and function. Cell Microbiol. 2011; 13(9): 1429–1439. PubMed Abstract | Publisher Full Text\n\nDong N, Zhu Y, Lu Q, et al.: Structurally distinct bacterial TBC-like GAPs link Arf GTPase to Rab1 inactivation to counteract host defenses. Cell. 2012; 150(5): 1029–1041. PubMed Abstract | Publisher Full Text\n\nSelyunin AS, Reddick LE, Weigele BA, et al.: Selective protection of an ARF1-GTP signaling axis by a bacterial scaffold induces bidirectional trafficking arrest. Cell Rep. 2014; 6(5): 878–891. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGermane KL, Spiller BW: Structural and functional studies indicate that the EPEC effector, EspG, directly binds p21-activated kinase. Biochemistry. 2011; 50(6): 917–919. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuttman JA, Samji FN, Li Y, et al.: Aquaporins contribute to diarrhoea caused by attaching and effacing bacterial pathogens. Cell Microbiol. 2007; 9(1): 131–141. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10659",
"date": "05 Oct 2015",
"name": "Pradeep Dudeja",
"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 first sentence conveys the wrong message. EPEC does not cause 1 million deaths but as quoted reference 1, it causes an average of 79000 dealth/year. The overall deaths due to diarrheal diseases are about 1 million/year. The authors should first outline the mechanisms of EPEC induced diarrhea including recent work on NaCl absorption than focus on tight junctions. The authors should refer to the recent papers on EPEC effects on tight junctions e.g. Glotfelty et al. Cell Microbiol. 2014",
"responses": [
{
"c_id": "1642",
"date": "04 May 2016",
"name": "Saima Aijaz",
"role": "Author Response",
"response": "Thank you for your comments Prof. Dudeja. We are working on addressing the issues raised by you and will incorporate them in the revised version."
},
{
"c_id": "1930",
"date": "18 Apr 2016",
"name": "Saima Aijaz",
"role": "Author Response",
"response": "Thank you for your valuable comments. I have now addressed the concerns you had and have incorporated the literature and references related to Cl- absorption as well as references by Glotfelty et al. I appreciate your comments as they have helped me to broaden the focus of this review and helped to highlight the importance of the disruption of ion channels and transporters by EPEC."
}
]
},
{
"id": "11552",
"date": "06 Jan 2016",
"name": "Fernando Navarro-Garcia",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting review, and I think it makes a positive review to the current knowledge about the effects of EPEC on the tight junctions. However, it will be interesting to note the observations posted by the first reviewer (Dr. Dudeja).",
"responses": [
{
"c_id": "1929",
"date": "18 Apr 2016",
"name": "Saima Aijaz",
"role": "Author Response",
"response": "Thank you for your comments. I have now amended the review to incorporate the suggestions of Prof. Dudeja."
}
]
}
] | 1
|
https://f1000research.com/articles/4-231
|
https://f1000research.com/articles/5-797/v1
|
03 May 16
|
{
"type": "Opinion Article",
"title": "The natural defense system and the normative self model",
"authors": [
"Philippe Kourilsky"
],
"abstract": "Infectious agents are not the only agressors, and the immune system is not the sole defender of the organism. In an enlarged perspective, the ‘normative self model’ postulates that a ‘natural defense system’ protects man and other complex organisms against the environmental and internal hazards of life, including infections and cancers. It involves multiple error detection and correction mechanisms that confer robustness to the body at all levels of its organization. According to the model, the self relies on a set of physiological norms, and NONself (meaning : Non Obedient to the Norms of the self) is anything ‘off-norms’. The natural defense system comprises a set of ‘civil defenses’ (to which all cells in organs and tissues contribute), and a ‘professional army ‘, made of a smaller set of mobile cells. Mobile and non mobile cells differ in their tuning abilities. Tuning extends the recognition capabilities of NONself by the mobile cells, which increase their defensive function. To prevent them to drift, which would compromise self/NONself discrimination, the more plastic mobile cells need to periodically refer to the more stable non mobile cells to keep within physiological standards.",
"keywords": [
"systems biology",
"complexity",
"physiology",
"robustness",
"immune system",
"quality control",
"network",
"self non self discrimination"
],
"content": "Introduction\n\nLiving organisms are protected by their immune system from infections. They are also threatened by the many errors which occur in their body. The ‘normative self model’ proposes that all abnormalities and disorders are taken care of by a unique system of natural defenses, which includes but exceeds the immune system1. After describing its composition and global architecture, I will show how it may protect the physiological self without attacking it. Overall, the theory, (which makes use of the tuning concept developed by Grossman and Paul2) is consistent and may help in understanding various physiological and pathological situations.\n\n\nThe hazards against which the organism must be protected\n\nExternal enemies come from the environment and mostly include infectious agents. Internal enemies, such as cancer cells, originate from the numerous mistakes which occur continually, within the body. They are often underestimated because most are corrected by quality control mechanisms. They include: errors in DNA replication, epigenetic alterations, incorrect transcription and splicing, errors in the synthesis3 and modification4 of proteins retrotransposon jumping5, unproper cellular migration, illegitimate cell pairing, organ dysfunction (such as, extracystoles in heart beats), etc.\n\nMost biological mechanisms involve a series of steps. Each has a small, but significant probability of error. Biological processes may be seen as a succession of trials and errors which converge towards the correct biological solution, under conditions which have been algorithmically formalized6. Each step has a certain yield, and a certain specificity. The combination of moderately specific events may produce an exquisitely specific output (though the final result may occasionally be incorrect). Such processes generate waste which must be recycled or eliminated (as in DNA replication and protein synthesis).\n\nError rates vary widely. The more steps a biological process involves, the more mistakes it is likely to make, even after error correction. Thus, the frequency of misincorporation of a nucleotide into replicating DNA is about 10-9; that of a wrong aminoacid into a protein (which involves many more steps) is higher than 10-5. Error rates would be higher, and unbearable for the organism, if it were not equipped with multiple quality control mechanisms. However, too many would consume too much time and/or energy. A ‘too close to perfect’ organism would not be competitive enough to survive. Therefore, mistakes are inevitable. The observed rates result from highly selected evolutionary trade-offs.\n\nAt a higher degree of organization, quality control mechanisms are found in the immune system, since (i) its own processes, (such as T lymphocyte selection) are quality controlled; (ii) it is itself a major quality control device, since it cures many unnoticed infections (revealed by antibodies in the serum of healthy people). It is likely that the same happens in at least certain cancers (as suggested, in particular, by the early dissemination of metastatic cells, which are kept silent by immune cells7). By extension, the natural defense system may cure many other adverse events (micro-bleeding, minor cardiovascular accidents, etc)8. However, this logical assumption awaits further experimental support, because error corrections are difficult to observe, unless sequellas remain after repair.\n\n\nThe architecture of the natural defense system\n\nIt is now accepted that the immune system does not only fight infectious agents9, it also intervenes in cancer10, so much so that several classical chemotherapeutic anti-tumor drugs (such as Temozolomide) have been shown to activate the immune system11. The belief that the adaptive system appeared once in evolution has been challenged by the finding that lamprey and hagfish have evolved an adaptive system which relies on molecules with LRR (leucin rich repeats) motifs, instead of the usual immunoglobulin fold12. Moreover, it was discovered recently that (like plants) certain bacterial species have evolved micro-RNA based adaptive immunity to destroy the genomes of infecting bacteriophages13. Finally, the borders of the immune system have been expanded, due to its functional relationships with the gut microbiota14 and the nervous system15. Thus, the limits of the immune system have changed several times.\n\nThe proposed ‘natural defense system’ considers everything that needs to be fixed in the organism. For example, neither clotting factors, nor piwi16, are considered part of the immune system. However, they do appear as bona fide defenses when one considers the potential damages generated by wounds or human genome destabilization by retrotransposons (including cancers17). Pain is a major warning system. Microbiota (in the gut and elsewhere), and mucus synthesis in the lung also help prevent infections. All contribute to defend the body against the hazards of life (Figure 1) and belong to the natural defense system. Because of its fuzzy borders, its size is hard to estimate. It must be significantly larger than the immune system, (at least a few % of human genes18). Arguably, adding up ‘cancer’ genes, plus some genes involved in the nervous, neuroendocrine and coagulation systems, might total 20% or more.\n\nAberrations and mistakes occur at all levels of the organism, and are listed on the left side. The major categories of quality control mechanisms are shown on the right. The immune system, (which makes up most of the ‘professional army’), covers one part. Intracellular quality controls, and mutual cellular surveillance (often by apoptosis) belong to ‘civil defenses’ which are individually exerted by all cells of the body (including those composing the professional army). Roads to diseases, and particularly to cancers rely on multiple aberrations that are not corrected by several of these quality controls.\n\nFrom now on, my use of military metaphores (common in immunology) should not obscure the preventive role of the natural defense system, which (more like a doctor than a soldier) solves many problems before they become pathological.\n\nThe body contains a vast majority of immobile cells, of which organs and tissues are made, and a minority of mobile cells, mostly found in blood and lymph. Others are motile; they reside in tissues and start moving upon stimulation (for simplicity, I will refer to them as ‘mobile’). Mobile cells exert a key defensive role, by moving to problematic site, (cf immune inflammation), where some proliferate, and/or function in destruction (eg cytolysis), or repair (clotting and healing). The ‘professional army’ of the organism, mostly recruited amongst these mobile cells, complements the ‘civil defenses’ provided by all cells, mobile and non mobile (which, for example, make interferons upon viral infection). Civil defenses include a large variety of intracellular and systemic quality control mechanisms (such as those involved in DNA replication, metabolism and temperature regulation). I will not discuss the idea that the human brain is a rupture innovation which allows man to manipulate its own natural defenses and invent new ones.\n\n\nThe internal consistency of the natural defense system\n\nEnemies from the outside and the inside are not fully distinguishable. Pathogens which penetrate inside the body are both recognized by their ‘foreign’ molecular motifs (such as bacterial lipopolysaccharide LPS), and by the disorders which they cause. The latter (such as death by necrosis instead of apoptosis, which delivers strong signals to the immune system), may be similar in infection, cancer and other pathological situations, so that the associated abnormalities may be detected and corrected by the same mechanisms. For example, the CD1 non-classical class I MHC molecules check on lipid metabolism by presenting particular lipids and metabolites to specific T cells, which in turn release cytokines in a variety of settings19,20. Reciprocally, tumors, as infecting pathogens, elicit specific antibodies and cytolytic T cells. Immune effector mechanisms (particularly macrophages) intervene in a variety of healing situations. Skin wounds call on coagulation devices as well as on immune cells and cytokines. Neuromediators are often linked to inflammation, chronobiology modulates defenses against infections, neuro-immuno-psychology starts making molecular sense21, and so on. Therefore, defense mechanisms often operate across various non physiological situations.\n\nThe continuum between the biology and the immunology of tumors. Tumor immunology has had its ups and downs, (immunology was absent from a seminal review on cancer written in 200022), but there is now overwhelming evidence that the immune system fights tumors. Correlatively, tumor immunotherapy is gaining ground, for example with the Chimeric Antigen Receptors (CAR) approach23. Of course, the immune system intervenes late, once several quality control mechanisms have failed. These include the intracellular control of mutations in pre-tumoral cells, and p53 related mechanisms which signal abnormalities, resulting in the apoptosis of the diseased cell, triggered either from within itself, or by neighbouring ones. It is only after a number of steps24, including progressive modifications of the tumor microenvironment25, that the immune system, with which it interacts26, takes action. In my view, this continuum of anti-tumor devices is better accommodated under the label of ‘natural defenses’, rather than sliced into at best overlapping segments as usually done.\n\nThe natural defense system intervenes at different levels to maintain the integrity of molecules (nucleic acids and proteins), individual cells, increasingly large sets of cells, up to organs, systems, and the entire body. Cancers of genetic origin involve these various levels, starting with the prevention of mutations up to organs. Infections skip the first round of genetic controls. The repair of an accidental, external, wound mobilizes the later stages. Multiple entries and exits are thus plugged on a general backbone, which links molecules to body parts and the entire body.\n\nThe set of natural defenses makes up a system. Natural defenses would not belong to a ‘system’ if they were a mere collection of disparate devices. Instead, they include many interconnected surveillance and correction mechanisms. In particular, apoptosis, although not usually presented as such, is a common quality control mechanism, which operates across the natural defense system. It is linked to the surveillance of many major intracellular failures; it is also related to mitochondria and to the utilization of energy27. It is a radical correction mechanism, when it comes to suppress a cellular problem that could not be solved. It operates both intra- and inter- cellularly, since healthy cells may deliver an apoptosis signal to sick ones.\n\nAbout complexity. Thanks to new technology and informatics, biological complexity can now be adressed more comprehensively, as illustrated by genome sequencing. However, as I discussed elsewhere28, one should not confuse ‘systematic’ and ‘systemic’ biology. The former documents complexity, without explaining it. As emphasized by S. Brenner29, the massive acquisition of ‘big data’ does not (and cannot) suffice to solve major biological problems. Instead, complexity requires specific conceptual tools.\n\nI now mention a few seminal papers. Engineers have dealt with complexity well before biologists. The notion of modularity was emphasized by Hartwell et al. (1999)30. Csete and Doyle (2002)31 have elaborated on a comparison between complex human artefacts (such as aircrafts) and biological systems. They showed that complexity in engineering and biology share the representation of objects by networks, the concept of an emergent property, and that of robustness. Later, Liu et al. (2011)32 used control theory to convincingly suggest that living systems are particularly complex, since it appears necessary to control the vast majority of their nodes (about 80%) to master the evolution of the biological networks analyzed (versus about 30% for the european electricity network).\n\nThe definition of robustness. In the 1860’s, Claude Bernard defined homeostasis as the ability of a system to maintain a balanced functioning despite outside constraints. Hence the well known quote: “The constancy of the internal environment is the condition for free and independent life: the mechanism that makes it possible is that which assured the maintenance, within the internal environment, of all the conditions necessary for the life of the elements”33. This concept strongly influenced cybernetics and engineering. Today, the notion of robustness (which some call ‘resilience’) adds to homeostasis, by equally considering the uncertainties associated with the « milieu intérieur » and the outside. Thus, Csete and Doyle (2002)32 define robustness as “the preservation of particular characteristics of a system despite uncertainties in components or the environment”. In a kind of pendulum’s swing, they make the point that it applies to biological systems. In my own words: “Robustness is a property that enables a complex system to keep on working, decently if not optimally, in spite of environmental hazards and internal failures” (the term ‘decently’ means that the system may go on working in a sub-optimal rather than optimal fashion).\n\nThe natural defense system confers robustness to the organism. Thus, robustness in engineering matches the role here assigned to the natural defense system. In other terms, the function of the latter is to confer robustness to the organism. This statement is important, because it provides the natural defense system with a unified function which makes evolutionary sense. Robustness is likely to be a major driver of evolution34 (at least within each species). In engineered systems, the space dedicated to robustness grows with time (the first aicrafts were much less robust than the current ones) and occupies more space. In living organisms, the number of « essential » genes35 is relatively low. A ‘minimal’ bacterium needs a few hundred genes, while E. coli has about 4 000. Therefore, more than 3 000 might be dedicated to robustness, including adaptative capabilities (for instance, using lactose instead of glucose).\n\n\nThe natural defense system is the guardian of physiology\n\nThanks to its surveillance, correction and repair capabilities, the natural defense system is the guardian of physiology, physiology being, in its medical sense, opposed to pathology. The term is philosophically and practically imprinted with the notion of normality, which is a basis of medical thinking36. The assessment of normality is heterogeneous in time, populations and cultures, without loosing its medical operational value.\n\nWhen identifying and fighting external and internal hazards, the natural defense system must not damage ‘the physiological self’ that is, “the physiological organism at all stages of its life”. This definition incorporates the dimension of time, since the body changes from childhood to adult and old age. The terms ‘physiology’ and ‘physiological’ refer to ‘norms’ and ‘normal’, which themselves concern molecular, cellular and multicellular structures and functions, and their way of responding to the environment.\n\nThe structural dimension. The physiological body is made of a vast number of structurally ‘normal’ molecules, cells and organs. Their catalogues are more and more comprehensive, though none is completed yet. New human genes, cell types, and even organs continue to be discovered. The gut microbiota is now considered a bona fide organ and the source of new metabolites37. Combinations of cell surface sugars are still being explored38. Thus, the composition and borders of the human body are still in question.\n\nThe numerous genetic polymorphisms further complicate the situation. For instance, major histocompatibility (MHC) class I molecules present a certain subset of self peptides to T cells. However, this ‘peptidic self’39 is almost unique to each person, because it is specified by the combination of MHC alleles borne by an individual. This feature partially accounts for MHC restriction and alloreactivity40. So, there cannot be a unique physiological catalogue of self structures shared by all. Polymorphisms also blur the notion of ‘normality’. For example, it may be difficult to define a ‘normal’ gene crowded with hundreds of polymorphisms, (and even more a ‘normal’ genome) without refering to its ‘normal’ function.\n\nThe functional dimension. After having adressed organic and systemic functions, physiology and pathology are now associated with cells and molecules. (for example, blood transaminases serve as an indicator of liver function). The genetic, epigenetic and environmental diversity, on top of functional fluctuations, broadens the standards of normality (cf. lymphocyte numbers and electrolyte concentration in a blood formula). The latter may reside in combinations of parameters, reflecting distinct states of biological networks. This is a trend in biomarkers research41.\n\nAt any time in life, the system must be ‘aware’ of the physiological standards, and make use of them to detect and correct defects, without damaging what works. In other terms, the physiological self is self-assessed. This statement is neither paradoxical nor tautological. It refers to the general paradigm of body development plans, of which the physiological self is a part. As for any development plan, it is somehow rooted in the genome, while exceeding by far the simplistic interpretation of a direct genetic control over its development and implementation.\n\nThe large corpus of physiological norms, and the complementary one of structural and functional abnormalities, grow rapidly, thanks to sustained biomedical research. Beyond the morphological norms (much improved by the progress of imaging), many norms deal with the molecular and/or cellular components of organs or systems, reflecting the activity of cell types, and the nature of cellular interactions. Therefore, many physiological norms lie in affinity and avidity constants which rule the interactions between molecules, molecules and cells and/or between cells (through their surface molecules). They are also part of the body plan.\n\nDefinition. What the natural defense system identifies and fights, therefore, is anything ‘off norms’, which I will refer to by the acronyme “NON-self”, meaning “Non obedient to the norms of the self”. The NON-self is thus defined by default. It includes everything abnormal, either ‘foreign’, or self. Like the physiological self, the NON-self has a structural arm (a mutated DNA sequence, bacterial LPS, a misfolded self protein, molecular patterns or aggregates with an unusual geometry, etc), and a functional one (aberrant metabolism, organ dysfunction, etc). Note that a ‘non physiological’ feature is only potentially (rather than necessarily) pathological, since, most of the time, it is corrected by the natural defense system.\n\nNON-self is mostly inhabited by chance. Most adverse events which threaten the physiological self are fortuitous. Even if their probability of occurrence is modulated by the environment and the ‘milieu intérieur’, most hazards of life are not deterministic. Infections, wounds, deleterious mutations and other internal mistakes, happen by chance. Furthermore, the number of possible hazards is huge, and some (such as an infection by a newly evolved pathogen) are unpredictable. Therefore, a major task of the natural defense system is to cope with chance. This feature profoundly imprints the mechanisms of discrimination between self and NON-self, which themselves exploit chance (as illustrated in the immune system by the random recombination of antibody and TCR gene segments and by the Darwinian process of antibody maturation in germinal centers).\n\nNON-self is not ignored, but dealt with. Infectious agents and cancer cells trigger specific actions against them. Similarly, repair activities are focused on the abnormal zones. NON-self is defined by default, but it is actively recognized. The famous, and much studied, problem of self/non self discrimination by the immune system, has to be translated into an issue of (physiological) self/NON-self discrimination by the natural defense system.\n\nBecause it is essential for survival, the natural defense system is necessarily robust. The same holds for self/NON-self discrimination, which must include several, possibly redundant, mechanisms, and various quality control devices (as well documented in the immune system). Furthermore, its enemies have been selected to be robust. Most pathogens have evolved very sophisticated escape mechanisms, without which they would be harmless. Poliovirus, HIV-1 and others have concentrated in their small genomes an amazing number of firewalls against the immune system. Similarly, cancers develop out of complex and elaborate selective processes, which yield cells that are robust enough to defeat the natural defense system. It may be expected that serious heart strokes, vascular failures and other pathologies occur after a long series of repaired defects, but this needs to be substantiated.\n\n\nThe normative self model\n\nPrevious theories of immune self/non self discrimination. The clonal deletion theory, first formulated in 1949 by Burnett, claimed that self-reactive immune cells are destroyed or inactivated. It was beautifully simple and dominated for decades, but did not account for the presence of numerous autoreactive B and T cells, and abundant self-reactive antibodies in blood and lymph. The resurrection of (suppressive) regulatory T cells42,43 added a well needed negative loop to explain major aspects of tolerance to self. The “danger theory”44 claimed that self constituents, if dangerous, can trigger an immune response (while non dangerous, non self constituents will not). The strengths and weaknesses of this approach have been discussed45. If clearly not generally applicable, it has emphasized the functional aspect of the physiological self (inasmuch as ‘danger’ is abnormal). The “discontinuity theory”46 has rightly emphasized the temporal dimension of immune stimuli and responses. Although hotly debated about adaptative immunity47, these issues have also be discussed in evolutionary contexts48, and about innate immunity38.\n\nDynamic tuning. The concepts underlying « dynamic tuning », proposed in 1992 by Grossman and Paul49, are best summarized by quotes taken from their 2015 review2. As it does “in other cell systems, neurons in particular, (…) dynamic tuning of cell responsiveness as the result of repeated stimuli, improves the ability of cells to distinguish physiologically meaningful signals from each other and from noise (…) eventhough the same sets of receptors may be utilized. In particular, lymphocyte activation thresholds are subject to tuning (…). Such tuning is also implicated in conferring flexibility to positive selection in the thymus, in controlling the magnitude of the immune response, and in generating memory cells. Additional functional properties are dynamically and differentially tuned in parallel via subthreshold contact interactions between developping or mature lymphocytes and self-antigen-presenting cells. (…). The built-in adjustability of intracellular control is utilized by the immune system to improve its organization and function”50. Importantly, each of these statements has received experimental support.\n\nThe three following points are most relevant for the current discussion:\n\n(i) cells may exist in a variety of states, even with the same sets of receptors, and this feature is a built-in property of their intracellular network;\n\n(ii) repeated stimulations help extracting meaningful signals out of environmental noise;\n\n(iii) subthreshold contact interactions between two cells may re-frame phenotypes (thus, in the periphery, transplanted mature naive CD8+ or CD4+ T lymphocytes retain their phenotype -without being activated- in congruent MHC+ mice, but loose it in MHC knock out mice). This type of self-recognition has been shown to promote the foreign antigen sensitivity of naive T cells51.\n\nThe maximization of their diversity helps actors of immunity to defeat chance. This major lesson derives from the diversity of antibodies and TCRs and the size of their repertoires. A large number of antibodies are generated at random. Affinity maturation increases their diversity, then their specificity. The exchange of constant regions (isotype switching) adds to diversity by promoting topological and functional redistributions of antibody repertoires. TCRs are also generated at random in large numbers. There is no TCR maturation; instead, T cells tune their reactivity in order to optimize their avidity for their partner (or target).\n\nRemarkably, immune responses can be elicited against just about anything. This holds for mice and men52, and in even for mutant mice with restricted repertoires which have (at least) 100 to 1000 times fewer distinct antibodies, B cells and T cells than humans53. Nevertheless, in all cases, the space of antigenic shapes is seemingly saturated. Therefore, adaptive responses are essentially scale free, implying that there is considerable potential for crossreactions between a given antibody or TCR and their molecular ligands40,54. This broad crossreactivity does not contradict the ‘exquisite’ specificity of the immune reactions, because specificity is raised to the appropriate level by additional mechanisms. As happens in T cells, tuning is a further means to broaden the diversity of phenotypes displayed by a given cell before adjusting its specificity.\n\nAll cells are auto-adaptable by tuning. I now postulate that all cells can adapt themselves and ajust their level of response according to their environment (this being a built-in property of all cellular networks). Grossman and Paul2 quote evidence of tuning in B cells, NK cells, eosinophils and dendritic cells. Beyond, tuning would apply to all cells, whether associated with immunity (cf. the plasticity of macrophages55,56), or not (fibroblasts, liver cells, and others).\n\nSpontaneous and/or induced fluctuations are necessary to expand the diversity of NON-self recognition. Thus, in order to cope with chance, the natural defense system needs to maximize its diversity of interactions. It does so by various means, one being to exploit the fluctuations of intracellular networks. This assumption is supported by the broad dispersion of gene transcription and expression observed in isolated, genetically identical, cells in the same environment57–59. A population of cells is, therefore, dynamically much more diverse than one might expect, and its ability to sense the environment much broader. Subsequently, the adaptative character of tuning through repeated stimulations is essential to extract meaningful signals out of environmental noise, and deal with them appropriately. There is experimental evidence that a given T cell which gives rise to a disparate progeny makes up a more robust immunity60. One may also hypothesize that tuning in innate cells helps understand why innate immunity alone protects many organisms so efficiently, despite the limited number of innate receptors.\n\nCells check upon themselves. In an isolated cell, all pathways leading to apoptosis are linked to the detection of mistakes that could not be corrected to a sufficient degree, or of deviations of the internal network that could not be re-balanced. The cell is programmed in such a way that it then commits suicide. Apoptosis is, for the isolated cell, the ultimate correction mechanism.\n\nCells double-check upon their neighbours. Cellular interactions are critical to maintain physiology, by ‘education’, selection or eradication. For example, in the periphery, subthreshold tuning based upon transient interactions with other cells is critical to maintain the properties of positively selected T lymphocytes. Apoptosis of sick cells triggered by normal ones is another type of check. Altogether, a moderately sick cell may correct itself, or be set back on track by a neighbour. If severely or irreversibly sick, it may commit suicide, or be killed by a neighbouring cell which induces its apoptosis or delivers lethal agents. Thus, cellular sickness is quality controlled from inside, and by neighbours.\n\nPhysiological norms concern the entire organism, but many lie at the cellular level. Intracellular norms mostly involve macromolecules, and translate into affinity constants (such as the affinity of a transcription factor for a promoter). The corresponding error detection and correction mechanisms pertain to the ‘civil defenses’. When by-passed, the cell becomes sick, and is taken care of by intra- and inter-cellular mechanisms. Higher scale disorders, in organs or the whole organism, are somehow referred back to individual cells (by lymphokines, hormones, metabolites, electric and other physical signals). Apart from extracellular microbes, the pathological objects to be discriminated are diseased cells (infected, tumoral or other). Thus, it makes sense to focus on cells and cellular norms.\n\nThe two types of auto-ajustable cells. (i) All cells undergo variations in their internal networks, and may, therefore, drift. However, those which are in constant contact with others in tissues and organs, are restricted in their capacity of tuning and cannot drift. If they did, they would die by apoptosis (either internal, or inflicted by their neighbours). This process is a basis of aging: tissues and organs are slowly empoverished, but not put in immediate danger, untill losses become too important (which stem cells may compensate). Cells belonging to tissues and organs constitute the « somatic self », which provides the necessary reference for the self-assessment of the physiological self. It follows the physiological variations of the organism over its life time.\n\n(ii) In contrast, mobile cells are not under permanent mutual control. They are in a ‘relaxed tuning’ state, and are susceptible to drift away from their standards. They need to be ‘reset’ by periodic encounters with cells of the somatic self (for example, a circulating lymphocyte will meet a a blood vessel epithelial cell more frequently than another lymphocyte). Thus, mobile cells, are allowed to fluctuate, but within certain limits.\n\n(iii) Both drift and reset may depend on the environment. The somatic self is likely not to be the same everywhere in the body and may differ in vessels, lymph nodes, or various tissues. For example, the same T cells may display distinct cytotoxic capabilities depending on their location, as suggested by the up-regulation of PD-1 in an allogeneic hematopoietic stem cell transplantation experiments61.\n\nThe professional army of the body is mostly recruited amongst mobile cells. The natural defense system thus combines civil defenses, disseminated in the entire body, with the services of a mobile professional army (which also has to defend itself, as any army does). Since hazards of life mostly happen by chance, the professional army relies on tuning to gain efficiency. However, it is constantly referred to the somatic self to keep in line with physiological standards (Figure 2).\n\nDiscrimination between the self and the NON-self. Discrimination is based on a simple principle: anything which lies off the norms is detected, corrected and/or destroyed, by civil defenses and/or the professional army. The fact that the latter needs ‘relaxed tuning’ to maximize its recognition capabilities creates a kind of no man’s land in the physiological self, a space where norms are less precise, and more opportunities given to crossreactions, mimicry, escape and so forth. Note that this grey zone may also be exploited by the NON-self. For example, tumor cells may use tuning to find their way out of host defenses. Infectious agents may do the same, thanks to so far unsuspected escape mechanisms.\n\nThe left side shows maximization of diversity of mobile cells (M) under the control of somatic cells (S). Thanks to tuning, various M cells (M1, M2, M3…) (in green, yellow and black) undergo diversification into (M1a, M1b…), (M2i, M2j …), (M3k, M3n…) (same colors with small variations), through deformations of their internal networks. S cells cannot diversify much because they are in close permanent contact. Diversified M cells (such as M1a, M3k, M3n) are occasionally reset to the M1 and M3 states by contact with S cells. The right side shows an aberrant cell (A) (in red), which is either altered (cancer) or infected. Thanks to network deformations (tuning), the population of mobile cells is more diverse, and there is a higher probability that a cell (M2i) properly recognizes the aberrant cell A. The recognition may be further improved by dynamic tuning which turns M2i into the more efficient M2ij, which will functionally adapt to deal with A.\n\n\nDiscussion\n\nThe proposed ‘natural defense system’ exceeds by far the immune system. Just as the latter, it is a pure mental construction, which I deem worthy of consideration for several reasons:\n\nIt unifies the fields of infectious diseases and internal pathologies. It provides a more comprehensive view of cancers, and facilitates the conceptual integration of immunology with other biological systems (for example, by linking microbiota and neurological diseases through inflammation).\n\nIt emphasizes the so far underestimated role of quality control and error correction mechanisms. Admittedly, it may seem difficult and/or artificial, to distinguish an error correction device from the primary biological mechanism when both are part of the same macromolecular complex. For example, in DNA replication, it is easier to identify mismatch repair enzymes than the proof-reading activity of DNA polymerase. Nevertheless, quality control and error correction mechanisms should be more systematically investigated. Note that their occasional failure may be pathological. Thus, certain manifestations of auto-immune diseases may be due to defective quality control rather than to primary defects.\n\nThe concept of robustness is fundamental. It provides a unified function to the natural defense system, which it links to evolutionary forces (its relationship with the concept of fitness would deserve attention).\n\nThe natural defense system, which provides robustness to the organism, must itself be robust. Its peculiar architecture, as depicted in this paper, has been selected to be so. It will be interesting to examine whether it resembles certain engineered systems34, and/or may inspire the design of new ones.\n\nThe overall scheme is relatively easy to communicate and teach. This is not a minor point, as immunology is rightly reputed to be abstruse for students and for the general public.\n\nThe two definitions of the self (the physiological organism at all stages of its life) and of the natural defense system are consistently bridged by the notion of physiology. The physiological self, in its structural and functional dimensions, relies on critical interactions between proteins and cells, some of which may be expressed as affinity and avidity constants, thresholds and/or windows. This set of physiological standards is particularly important for mobile cells (such as T cells).\n\nThe model is based on (a) physiological norms, and (b) their verification by an adequately structured control and defense system.\n\nPhysiological norms are somehow written in the genome. Their implementation during development, as well as their maintenance during life, rely on the body plans of the natural defense system, which should be further explored62. The set of norms must itself be robust with respect to the numerous genetic and epigenetic changes which occur in the body. In humans, the rate of somatic mutations (10-8/10-9) and the number of cell divisions from germ line to adult (by the hundreds for spermatozoa) are high enough to generate many gametes bearing multiple mutations. The latter should be viewed as impacting their internal networks, rather than single genes or simple functions. A network adapted genetic thinking is thus needed. I have earlier speculated that the ‘domestic’ part of body plans may undergo selection at checkpoints during gametogenesis63.\n\nThe verification of norms is the duty of an army which relies on two categories of cells: the non mobile, aggregated ones, which make up the somatic self (where most civil defenses lie), and the mobile ones (which make up the core of the professional army). The latter have to periodically refer to the somatic self to keep in line with the physiological standards.\n\nComments - The tuning-based extension of recognition capabilities of mobile cells might generate uncontrolled drift, and the natural defense system would not be robust enough, if mobile cells were not checked by immobile cells. This also provides a means by which the set of mobile cells adapts to age.\n\n- The generalization to all cell types of the notion of tuning, initially elaborated for T lymphocytes, might be further documented by analyzing the different states of intracellular networks in single cells of various types. The ‘elasticity’ of cellular networks (a particular kind of epigenetic phenomenon) might be modeled by introducing the notion of entropy at the single cell level, following Shannon’s theory of information64,65.\n\n- Current technologies66,67 allow to study the interactions of a single mobile cell with an immobile layer of cells, mimicking a tissue. The model predicts a change in the internal networks distribution of the single cell (a reduction of the cellular entropy).\n\n- It would be important to define the physiological borders of the relaxed tuning state. One may speculate that the internal networks of cells in a relaxed tuning state display an elastic behaviour, which would periodically drive them back to average. Even so, it would be surprising that the borders are not surveyed at certain checkpoints involving: Fas and Fas ligand (for apoptosis), MHC expression (for NK cells), cell surface markers such as CD59 (as inhibitors of complement), and possibly others as yet undiscovered.\n\n- It will be critical to evaluate (in vitro, and preferably in vivo) the time which elapses between two effective checks. This parameter is key, because it defines the time window during which interventions can be made. Repeating interventions at proper times may thus lead to desensitization, as already recognized and discussed2,45.\n\n- Immune changes with time, (particularly aging), have been mostly analyzed at the level of the components of the immune system. Additional studies might relate changes in natural defenses with the physiological evolution of the somatic self. This may help understand pathological variations in relation with age.\n\nThese above considerations support a very dynamic view of physiology, which results from the realization that, in Claude Bernard’s milieu intérieur, the elements are themselves uncertain, and, to some extent, renewable. Current research promotes a vision of our body that evokes the apologue of the Delphies’ boat, which seemingly remains the same, while most of its constituents have been changed. Our body does so by itself (though now complemented by medicine), with 70 billions cells renewed every day.\n\nWe are largely unaware of these uncessant internal fights and of the remarkable robustness of our organism. It may be more comfortable to ignore that our natural defense system permanently prevents and/or cures many infections, cancers, cardiovascular disorders, and so on. Nevertheless, understanding, then mastering better, these physiological dynamics, which maintain a stability slowly destroyed by physiological aging, will ultimately help improve our health.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author declares that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nI thank Philippe Bousso and Paola Castagnoli for stimulating discussions, Denise Derhy and Kiri Crouchman for her help, Christian Bréchot and the Pasteur Institute for overall support. I am grateful to College de France, the havre of liberty where I could develop in my teaching most of the ideas exposed in this paper.\n\n\nReferences\n\nKourilsky P: Le jeu du hasard et de la complexité, la nouvelle science de l’immunologie. Odile Jacob, Paris, 2014. Reference Source\n\nGrossman Z, Paul WE: Dynamic tuning of lymphocytes: physiological basis, mechanisms, and function. Annu Rev Immunol. 2015; 33: 677–713. PubMed Abstract | Publisher Full Text\n\nde la Cruz J, Karbstein K, Woolford JL Jr: Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo. 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}
|
[
{
"id": "14013",
"date": "26 May 2016",
"name": "Bernard Malissen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 opinion article by P. Kourilsky proposes a new model of the immune system called the «normative self model». It is vividly written and discusses several former models - such as the one promoted by Paul and Grossman - and it also capitalizes on recent experimental findings. It will be interesting to add a couple of sentences on innate lymphoid cells, unconventional T cells (e.g. MAIT, gd T cells, and CD8aa+ IELs), and tissue-resident memory T (TRM) cells since these diverse cell types play a role in the preservation of tissue integrity and function during homeostasis, infection, and non-infectious perturbations. More importantly, the aim of a model is to provide experiments that can be tested and clearly refuted or validated. Therefore, I will strongly encourage P. Kourilsky to include a section in which a couple of such experiments are suggested.",
"responses": []
},
{
"id": "13692",
"date": "27 May 2016",
"name": "David H. Margulies",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this opinion article, Kourilsky articulates a sweeping, holistic perspective on the plethora of innate defense mechanisms that exist at the molecular, cellular, tissue, organ, and organismal levels to ensure survival. The author broadens the definition of innate defenses to include not only cells traditionally associated with immunity such as macrophages and dendritic cells, but rather each and every cell and organ system in the organism.\nThe division of function is between cells that are sessile, such as those organized into tissues and organs, and that are motile such as those of the hematopoetic system. A key idea is the “normative self” – a homeostatic physiological state to which both types of cells contribute. The normative self is learned and is continuously reinforced by interactions between sessile and motile cells that tune the system to respond appropriately to neoplastic cells, aberrant inflammatory responses, and environmental perturbations such as infectious disease. The essay is thought-provoking and attempts, successfully in our opinion, to highlight the landscape of adaptable interactions that is necessary to sustain life. Conceivably, this perspective may be applied beyond the individual organism to the interplay of communities and even higher order populations.",
"responses": []
}
] | 1
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https://f1000research.com/articles/5-797
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https://f1000research.com/articles/5-787/v1
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29 Apr 16
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{
"type": "Review",
"title": "Recent advances in echocardiography: strain and strain rate imaging",
"authors": [
"Oana Mirea",
"Jurgen Duchenne",
"Jens-Uwe Voigt",
"Oana Mirea",
"Jurgen Duchenne"
],
"abstract": "Deformation imaging by echocardiography is a well-established research tool which has been gaining interest from clinical cardiologists since the introduction of speckle tracking. Post-processing of echo images to analyze deformation has become readily available at the fingertips of the user. New parameters such as global longitudinal strain have been shown to provide added diagnostic value, and ongoing efforts of the imaging societies and industry aimed at harmonizing methods will improve the technique further. This review focuses on recent advances in the field of echocardiographic strain and strain rate imaging, and provides an overview on its current and potential future clinical applications.",
"keywords": [
"echocardiography",
"strain rate imaging",
"echocardiographic strain",
"speckle tracking"
],
"content": "Introduction\n\nFor decades, two-dimensional (2D) and Doppler echocardiography were the central pillars of evaluating left ventricular (LV) function. For purely practical reasons, many clinicians still resort to measurements of LV ejection fraction (EF) as well as the visual analysis of myocardial wall motion when they evaluate LV global and regional performance. However, these methods have a significant inter-observer variability as they depend on the skills and experience of the user. The undisputed need for simple, readily accessible and reliable methods for evaluating function has driven industry towards the development of semi- or fully- automated methods and post-processing tools for quantifying LV function.\n\nStarting from first experiences with tissue Doppler, velocity imaging was followed by deformation imaging. But with the introduction of speckle tracking for analyzing images, quantitative analysis of myocardial function by deformation imaging had its breakthrough into clinical echocardiography. The recent recommendations released by the European Association of Cardiovascular Imaging (EACVI) and the American Society of Echocardiography (ASE) acknowledged the additional value of deformation measurements over traditional functional parameters, such as LV EF, and recommended the technique now for clinical use1. This review will discuss the most recent developments in the field of strain imaging and the application of the method in the clinical setting.\n\n\nWhat is strain?\n\nStrain is defined as the fractional change in length of a myocardial segment relative to its baseline length, and it is expressed as a percentage. Strain rate is the temporal derivative of strain, and it provides information on the speed at which the deformation occurs. Strain is a vector and the complete description of the complex deformation of a piece of myocardium requires three normal and six shear strain components. For practical reasons, the normal strains which are preferred for clinical use are oriented along the coordinate system of the LV; they describe radial thickening and thinning as well as circumferential and longitudinal shortening and lengthening. Lengthening or thickening of the myocardium is represented by positive strain values, whereas negative values represent shortening or thinning. The most commonly used parameter is longitudinal strain, which can be expected to be around 20% in all regions of the LV2.\n\nStrain is ideally suited to quantify myocardial function regionally, but with the introduction of speckle tracking, a new parameter for global LV function assessment called “global strain” has been introduced. In the longitudinal direction, global longitudinal strain reflects the deformation along the entire LV wall which is visible in an apical image. The measurements from all three apical views are combined to give an average GLS value.\n\nIt must be noted that myocardial deformation is load-dependent. Therefore, strain and strain rate measurements must be interpreted considering ventricular wall thickness and shape as well as pre- and after-load.\n\n\nHow to measure strain?\n\nTissue Doppler Imaging (TDI) was the first method used for directly measuring myocardial deformation by echocardiography. Since a regional velocity gradient is analytically identical with the temporal derivative of a change in length, strain rate can be directly calculated from two velocity samples at a known distance apart. Integrating strain rate over time results in strain3. The method is well validated4 and has been shown to provide valuable data in a wide range of conditions5. It benefits particularly from the high frame rate of echocardiographic TDI and therefore is the method of choice in all situations where short-lived mechanical events and fast changes in deformation (e.g., in diastole) have to be measured.\n\nJust over a decade ago, speckle-tracking echocardiography (STE) was proposed6,7 and validated8–10 as an alternative tool for measuring myocardial function. Observations in large patient populations showed encouraging results regarding its applicability8,11 in the clinical setting. The tracking algorithm identifies specific myocardial patterns (commonly named “speckles” or “features”) on conventional B-mode echocardiographic images and follows the motion of these patterns frame-by-frame. The potential to track the speckles in any direction within a 2D image allows the calculation of myocardial velocities, displacement, strain and strain rate in any given direction. This multidirectional tracking ability along with its angle independency12 are often regarded as major advantages over TDI. However, STE has also been shown to depend significantly on good image quality and proper image geometry. Because speckle tracking is derived from grey-scale images which have lower frame rates than TDI, measurements of motion and deformation are most reliable for events that last longer, such as systole. Tracking-based measurements of velocity and rate of deformation, however, should only be attempted with caution. The fast and user-friendly (semi-)automated post-processing is the biggest advantage of the technique.\n\nIn theory, tracking can be performed in all three dimensions when three-dimensional (3D) echocardiographic image data are available. However, the problems of frame rate and image quality as explained for 2D tracking are potentiated in 3D, while the advantage of a fully three-dimensional deformation analysis remains a hypothesis. Therefore, 3D STE must currently be regarded as an experimental method13.\n\n\nDefinitions and conventions\n\nTo achieve reproducible measurements, the method of measuring strain needs to be defined and communicated together with the result. The definition comprises not only the interrogated deformation component (longitudinal, circumferential, or radial) but also the sampling position within the myocardium (e.g., endocardial, midwall, or full wall) and the temporal definition of the measured parameter, as conventions are lacking in this field and the definitions are dependent on the vendor of the analysis software. In particular, definitions of timing can influence the measurement without being noticed by the user.\n\nMyocardial deformation is a cyclical process, and the definition of when in this cycle the myocardium can be assumed to have reached its “baseline length” is completely arbitrary. However, it is extremely relevant for defining what “zero strain” is. In physiology lessons, we are taught to consider end-diastole as the reference point in the cardiac cycle, but this is a difficult definition as it requires the mitral valve closure to be measured. Therefore, most strain analysis softwares use surrogate parameters such as the R-peak of the QRS complex in the electrocardiogram. The time of the R-peak, however, can deviate considerably from the true mitral valve closure time, in particular when conduction delays are present14.\n\nA similar question occurs when it comes to the definition of end-systole. In physiology, we use the time of aortic valve closure. In strain software, however, several vendors use the nadir of the global strain or volume curve as an approximation, since it can be calculated easily from the tracking data. Again, particularly in conduction delays or regional dysfunction, this surrogate can be very wrong14. Therefore, a good software should allow the measurement of aortic valve closure and its implementation in the strain analysis.\n\nFigure 1 displays the impact of timing changes on strain measurements. Figure 2 shows an overview of commonly used definitions, such as peak systolic strain, end-systolic strain, post-systolic strain (PSS), and peak strain. As can be seen, the definition of end-systole is of particular interest, as derived parameters such as post-systolic shortening directly depend on it.\n\nA) Speckle tracking longitudinal strain in a dilated ventricle with left bundle branch block. The yellow and blue dot indicate the origin of the strain curves of the same colour in the lower panels, where in addition a dotted white curve represents global longitudinal strain. (B) Correct definition of end-diastole (ED) and end-systole (ES). (C) The definition of ES has been moved by + 4 frames (green arrow indicates initial ES; green dashed line indicates current ES). Note the impact on the measured systolic strain value. (D) Here, the definition of ED has been moved by + 4 frames (white arrow indicates initial ED; white dashed line indicates current ED). Also, in this case, the measurement of end-systolic strain is indirectly affected due to a shift in baseline.\n\nPeak systolic strain (SS) is always measured before aortic valve closure (AVC). End-systolic strain (ES) is measured on AVC. Post-systolic strain (PSS) peaks after AVC.\n\nIn recent years, functional imaging based on speckle tracking has entered the clinical arena. Hundreds of publications have explored the potential of STE to improve the prognostic and diagnostic accuracy of echocardiography in a wide variety of cardiac pathologies associated with LV dysfunction.\n\nIn the general population, lower GLS was shown to be a powerful and independent predictor for vascular events such as stroke and myocardial infarction and for new onset of atrial fibrillation15,16. GLS also demonstrated prognostic value for adverse outcomes in patients with heart failure17–19, coronary artery disease (CAD)20–22, valvular heart disease23,24, and cardiomyopathies25.\n\nFurthermore, GLS proved to be a superior predictor of all-cause mortality when compared with LV EF and myocardial wall motion in patients with CAD26,27 or chronic kidney disease28. Longitudinal strain measurements also showed encouraging results in identifying early LV impairment in patients undergoing chemotherapy29,30 and in subjects with chronic nephropathy31 or diabetes mellitus32.\n\nPossibly the most valuable clinical application of strain measurements is the evaluation of regional dysfunction in patients with CAD. Early experimental validation against sonomicrometry showed that both Doppler-derived4 and STE-derived33 longitudinal strain can detect the presence of ischemia. Additionally, encouraging data were reported with respect to the ability of strain measurements to predict the extent of the ischemic area34 and to differentiate between transmural and non-transmural scar35.\n\nPSS, defined as the presence of regional myocardial shortening after aortic valve closure, is considered a hallmark for myocardial ischemia (Figure 3). The presence of PSS in areas with ischemic insult has been demonstrated in early studies by using TDI-derived measurements36,37. A longer persistence of the PSS after an ischemic event was associated with more severe coronary obstruction38. Nevertheless, it is important to consider that although PSS is a very sensitive marker of regional dysfunction, it is never specific for a certain pathology; PSS always needs to be interpreted in a clinical or pathophysiological context or both. PSS at rest can be a sign of ischemia, myocardial scar, or other conditions36. PSS which occurs during stress echocardiography, however, is very likely to be caused by ischemia38, so it can improve the accuracy of detecting CAD during a dobutamine stress test39. Measurements of longitudinal strain during dobutamine stress echocardiography for detecting CAD were reported to be feasible in 75–100%40,41. Whether these numbers are realistic in a routine clinical scenario, however, remains doubtful.\n\n(A) Segmental longitudinal strain curves in an apical two-chamber view of a patient with inferior infarction. The bold yellow curve is derived from the infarcted inferobasal segment. Note the pronounced post-systolic shortening (arrow). (B) Cardiac magnetic resonance imaging with delayed enhancement of the same patient. A scar is present in the basal inferior region (arrows). Abbreviations: AVC, aortic valve closure; GS, global strain.\n\nStrain imaging allows the assessment of asynchronous LV deformation (e.g., by measuring the time to peak strain). Haugaa et al. demonstrated in patients with CAD that abnormalities in synchronicity, referred to as mechanical dispersion and defined as the standard deviation of the time to peak regional shortening, could identify those with high risk for arrhythmias42. An example of larger mechanical dispersion due to the presence of ischemia when compared with normal myocardium is presented in Figure 4.\n\n(A) Normal heart. All strain curves peak around aortic valve closure (AVC). (B) Infarct patient with inducible arrhythmias in the electrophysiology lab. Note the wide dispersion of the segmental peak strains. Abbreviations: GS, global strain.\n\nIn past years, many indices have been suggested to identify potential responders to cardiac resynchronization therapy (CRT). Initial parameters were based on time-to-peak velocity measurements and could successfully detect dyssynchrony but failed to show added predictive value in prospective clinical trials43–48. The reason can be found in the inability of velocity measurements to distinguish wall motion due to contraction and wall motion due to tethering which prevents conclusive description of the sequence of wall activation. Later, deformation-based parameters have been suggested, focusing on the timing difference measured between dyssynchronous walls49–54. Such parameters are potentially useful but only if they identify signatures in the myocardial deformation pattern which are specific for hearts amenable to CRT. More recently, a new index has been proposed which is based on the non-invasive assessment of regional myocardial work and which combines regional LV deformation with an estimate of LV pressure55,56. It remains to be determined whether such parameters will prove to be superior to the more easy, direct visual or quantitative evaluation of the characteristic motion patterns of dyssynchronous hearts57–59. Both the fast early systolic inward motion and the subsequent stretching of the septum (septal flash), as well as the rocking motion of the LV apex (apical rocking), can be directly assessed and have been shown to successfully predict CRT response in a clinical setting with both a qualitative and a quantitative approach60.\n\nRecent research has demonstrated the complementary character of GLS and EF. Both parameters describe global LV function, but both do it in a different way. Although the parameters follow a linear fit of EF=3|GLS| in most situations61, GLS and EF may diverge depending on the underlying pathology, which could offer added diagnostic information. Several studies have shown that GLS is more sensitive to subtle changes in myocardial function which, for example, could be used in the follow-up of patients receiving chemotherapy62,63. In hypertrophic pathology, GLS is frequently reduced while EF is still normal64. A higher EF/GLS ratio was found to differentiate cardiac amyloidosis from other pathologies with increased LV wall thickness, such as hypertrophic cardiomyopathy65.\n\n\nAdvances in the standardization of strain measurements\n\nEstablishing the reliability of STE is a prerequisite to its clinical implementation. Although STE has proved to have numerous advantages, such as large availability, high feasibility, and added clinical information, there are still debates regarding the potential discrepancy of strain measurements between vendors66,67.\n\nIn recognition of this problem, the EACVI has initiated a task force together with the ASE and industry partners. The aims of this task force are to identify and minimize sources of variability between strain measurements and to standardize definitions for strain measurements3,68. The first results of the initiative provide insights on different speckle tracking algorithms, current terminology, and specific technical issues such as image acquisition or timing of measurements3.\n\nIn a first comparative study between all major vendors of echocardiography machines and strain software, GLS measurements have been found to show considerable inter-vendor differences, whereas its reproducibility was consistently comparable to or even better than that of conventional echocardiographic parameters69. These findings imply that GLS measurements can be considered a reliable tool in the clinical routine as long as repetitive measurements are performed with the same equipment. On the contrary, the comparison of data obtained with different post-processing software should be avoided.\n\n\nFuture perspectives\n\nThe assessment of LV regional function by speckle tracking has not yet been intensively tested. In particular, vendor-specific differences in the tracking algorithms, such as the number and dimension of kernels, regional smoothing, underlying models of LV motion, or others, may account for more significant differences at a segmental level than at a global level.\n\nMoreover, since regional function can be interpreted through various parameters, the most reproducible and robust for the definition of regional dysfunction remains to be determined. Given the potential value of regional strain measurements, the ongoing efforts of the EACVI/ASE Task Force focus on identifying reasons for the variability of regional strain parameters between vendor-specific software. Positive results would be a remarkable step forward in the process of endorsing regional strain measurements.\n\n\nConclusions\n\nEchocardiographic deformation imaging has developed into an indispensable tool for the clinical assessment of a wide range of cardiac pathologies, and current guidelines recommend its use because of its feasibility and robustness. However, one should remain aware of the pitfalls which still exist and which are easily overlooked because of the user-friendly one-click-gives-it-all approach of most software solutions. Joint efforts of echocardiography associations and industry partners to resolve these pitfalls, to minimize inter-vendor differences, and to standardize measurement definitions will provide a solid valid basis for a widespread use of the technique in the clinical routine.\n\n\nAbbreviations\n\n2D, two-dimensional; 3D, three-dimensional; ASE, American Society of Echocardiography; CAD, coronary artery disease; CRT, cardiac resynchronization therapy; EACVI, European Association of Cardiovascular Imaging; EF, ejection fraction; GLS, global longitudinal strain; LV, left ventricular; PSS, post-systolic strain; STE, speckle-tracking echocardiography; TDI, tissue Doppler imaging.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nLang RM, Badano LP, Mor-Avi V, et al.: Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging. Eur Heart J Cardiovasc Imaging. 2015; 16(3): 233–70. PubMed Abstract | Publisher Full Text\n\nYingchoncharoen T, Agarwal S, Popović ZB, et al.: Normal ranges of left ventricular strain: a meta-analysis. J Am Soc Echocardiogr. 2013; 26(2): 185–91. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nVoigt JU, Pedrizzetti G, Lysyansky P, et al.: Definitions for a common standard for 2D speckle tracking echocardiography: consensus document of the EACVI/ASE/Industry Task Force to standardize deformation imaging. 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PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLeenders GE, Lumens J, Cramer MJ, et al.: Septal deformation patterns delineate mechanical dyssynchrony and regional differences in contractility: analysis of patient data using a computer model. Circ Heart Fail. 2012; 5(1): 87–96. PubMed Abstract | Publisher Full Text\n\nSzulik M, Tillekaerts M, Vangeel V, et al.: Assessment of apical rocking: a new, integrative approach for selection of candidates for cardiac resynchronization therapy. Eur J Echocardiogr. 2010; 11(10): 863–9. PubMed Abstract | Publisher Full Text\n\nStankovic I, Aarones M, Smith HJ, et al.: Dynamic relationship of left-ventricular dyssynchrony and contractile reserve in patients undergoing cardiac resynchronization therapy. Eur Heart J. 2014; 35(1): 48–55. PubMed Abstract | Publisher Full Text\n\nStankovic I, Prinz C, Ciarka A, et al.: Relationship of visually assessed apical rocking and septal flash to response and long-term survival following cardiac resynchronization therapy (PREDICT-CRT). Eur Heart J Cardiovasc Imaging. 2016; 17(3): 262–9. PubMed Abstract | Publisher Full Text\n\nPedrizzetti G, Mangual J, Tonti G: On the geometrical relationship between global longitudinal strain and ejection fraction in the evaluation of cardiac contraction. J Biomech. 2014; 47(3): 746–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nThavendiranathan P, Poulin F, Lim KD, et al.: Use of myocardial strain imaging by echocardiography for the early detection of cardiotoxicity in patients during and after cancer chemotherapy: a systematic review. J Am Coll Cardiol. 2014; 63(25 Pt A): 2751–68. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFlorescu M, Magda LS, Enescu OA, et al.: Early detection of epirubicin-induced cardiotoxicity in patients with breast cancer. J Am Soc Echocardiogr. 2014; 27(1): 83–92. PubMed Abstract | Publisher Full Text\n\nOzawa K, Funabashi N, Takaoka H, et al.: Characteristic myocardial strain identified in hypertrophic cardiomyopathy subjects with preserved left ventricular ejection fraction using a novel multi-layer transthoracic echocardiography technique. Int J Cardiol. 2015; 184: 237–43. PubMed Abstract | Publisher Full Text\n\nPagourelias ED, Duchenne J, Mirea O, et al.: The Relation of Ejection Fraction and Global Longitudinal Strain in Amyloidosis: Implications for Differential Diagnosis. JACC Cardiovasc Imaging. 2016; pii: S1936-878X(15)00981-X. PubMed Abstract | Publisher Full Text\n\nRisum N, Ali S, Olsen NT, et al.: Variability of global left ventricular deformation analysis using vendor dependent and independent two-dimensional speckle-tracking software in adults. J Am Soc Echocardiogr. 2012; 25(11): 1195–203. PubMed Abstract | Publisher Full Text\n\nBansal M, Cho GY, Chan J, et al.: Feasibility and accuracy of different techniques of two-dimensional speckle based strain and validation with harmonic phase magnetic resonance imaging. J Am Soc Echocardiogr. 2008; 21(12): 1318–25. PubMed Abstract | Publisher Full Text\n\nThomas JD, Badano LP: EACVI-ASE-industry initiative to standardize deformation imaging: a brief update from the co-chairs. Eur Heart J Cardiovasc Imaging. 2013; 14(11): 1039–40. PubMed Abstract | Publisher Full Text\n\nFarsalinos KE, Daraban AM, Ünlü S, et al.: Head-to-Head Comparison of Global Longitudinal Strain Measurements among Nine Different Vendors: The EACVI/ASE Inter-Vendor Comparison Study. J Am Soc Echocardiogr. 2015; 28(10): 1171–1181, e2. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "13662",
"date": "29 Apr 2016",
"name": "Giovanni Di Salvo",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13663",
"date": "29 Apr 2016",
"name": "Tomasz Kukulski",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13664",
"date": "29 Apr 2016",
"name": "Alan Fraser",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-787
|
https://f1000research.com/articles/5-785/v1
|
29 Apr 16
|
{
"type": "Review",
"title": "Geroscience approaches to increase healthspan and slow aging",
"authors": [
"Simon Melov"
],
"abstract": "For decades, researchers in the biology of aging have focused on defining mechanisms that modulate aging by primarily studying a single metric, sometimes described as the “gold standard” lifespan. Increasingly, geroscience research is turning towards defining functional domains of aging such as the cardiovascular system, skeletal integrity, and metabolic health as being a more direct route to understand why tissues decline in function with age. Each model used in aging research has strengths and weaknesses, yet we know surprisingly little about how critical tissues decline in health with increasing age. Here I discuss popular model systems used in geroscience research and their utility as possible tools in preclinical studies in aging.",
"keywords": [
"Geroscience",
"healthspan",
"slow aging",
"lifespan",
"longevity"
],
"content": "Introduction\n\nWe are at a tipping point in the biology of aging—from lifespan extension per se to maintaining and extending health in late life. Since the early 1980’s, there have been serious efforts to use genetic approaches to extend lifespan in model systems such as Caenorhabditis elegans1–6, Drosophila7–15, and, increasingly, mice16,17. Collectively, such efforts fall under the catch-all term “geroscience”, which describes interdisciplinary efforts to better understand the biology of aging with a view towards improving healthcare in the elderly18. Recently, the tried and true genetic approaches of the 1990’s and early 2000’s in geroscience research have been increasingly giving way to a plethora of pharmacological approaches to extend lifespan. This has been in conjunction with efforts to simultaneously increase healthspan19–28, thereby providing a preclinical rationale for similar studies in human beings.\n\nIt has been reported that lifespan and healthspan can be extended in invertebrates using a variety of pharmacological approaches, including single antioxidants through small molecule screens and natural compounds23 as well as some anticonvulsants29. Not to be outdone, there are also supporting data for lifespan/healthspan extension in mice using repurposed US Food and Drug Administration (FDA)-approved drugs, novel chemical compounds, and biologicals (Table 1). Before examining key concepts in geroscience that drive a lot of the excitement in the pharmacology of lifespan/healthspan extension, it is necessary to first of all define what we mean by aging and healthspan. This is particularly germane in the model systems most commonly used in the biology of aging. By no means is the definition of such terms straightforward, and eminent figures in the field have spent considerable effort clarifying such apparently simple concepts. Caleb Finch of USC in his highly respected tome Longevity, Senescence, and the Genome30 devoted several chapters towards defining what is meant by “aging”—or, as he prefers to denote it, “senescence”. More recently, similar efforts to define aging/senescence have been discussed at length by several other investigators31–34. Some popular definitions of aging in a geroscience context have included the following:\n\nuse of mortality kinetics of an aging population to derive a mathematical definition\n\nthe length of life after the reproductive period\n\nthe probability of death with increasing age\n\nFor the purposes of this article, the term “aging” refers to post-reproductive changes that adversely affect lifespan. However, to define healthspan in the context of geroscience is perhaps even more difficult.\n\nIndependent Replication refers to whether or not an independent group replicated the original reported result. X refers to failure to replicate, while a check mark indicates the key finding was replicated. Challenge Publication lists the reference where the independent group confirmed or failed to replicate key aspects of the initial report.\n\nHealthspan is commonly interpreted to mean “maintenance of functional health with increasing age”. By necessity, this means one has to understand what it is to be healthy for multiple different systems and tissues. In human beings, this is perhaps non-controversial—one can access high-quality data collected from many thousands of individuals of both sexes as well as differing ethnicities while controlling for multiple lifestyles. One can then establish age-dependent measures for many different aspects of human biology35–41. These include measures of cardiovascular and cognitive function, movement (walking speed), renal function, and hemodynamic function, to name a few. Typically, such functional measures peak in early adulthood, then decline at different trajectories as the individual ages42. There are many factors that can modulate the slope of such a functional decline with age, including exercise, diet, and lifestyle. Maintaining function and independence with age using selective and specific interventions is arguably the single biggest challenge currently facing geroscience. For the model systems commonly employed in the study of aging biology, identifying functional measures that are relevant to human healthspan is quite difficult. In nearly all model systems used in the biology of aging, healthspan measures have been collected from aging animals not necessarily because of their relevance to human aging but because methods exist that allow one to measure the metric in question over time. Amongst these metrics, there is one clear measure that is very well established as being a robust biomarker of healthspan in human aging, and that is the measurement of movement with age43–45. A sound argument can be made for measuring this parameter in model systems of aging to ensure potential translational relevance.\n\n\nIt’s all about the movement!\n\nFor some time, it has been known that movement, especially walking speed, is correlated with increased longevity and a reduction in morbidity in human beings46,47. Movement is perhaps the simplest metric to measure as a functional output of age. Despite its apparent simplicity, walking is a highly complex task, which integrates many different systems including balance, strength, cognitive function, and multiple senses. Walking speed is therefore an integrative physiological outcome, which may be why it has been so tightly linked to the maintenance of health in the elderly. A reasonable extrapolation is therefore to understand the relationship between overall activity and aging pathophysiology. There are large-scale efforts underway to better understand how activity levels modulate longevity, resistance to disease, and function in human beings using personalized tracking devices such as the Fitbit, Apple Watch, or similar devices. Arguably, we should have a deep understanding of how activity levels modulate aging and health in model systems due to our complete control over the environment and genetics. In addition, the economics of carrying out such studies in models are far more practical for obvious reasons.\n\n\nMovement as a healthspan metric in model systems\n\nUnfortunately, the literature is hardly replete with such studies. In fact, we are in the infancy of beginning to understand how activity modulates healthspan in model systems. There have been sporadic reports correlating a decline in movement with age for more than 30 years in diverse model systems of aging48–58. These studies typically use a variety of different approaches to relate movement rates with age or with measures such as gene expression or some other “omic” outcome. There are comparatively few reports in which objective measures of movement rate have been taken, particularly with regard to high-resolution temporal density. Another area not commonly studied is the capture of individual variation with movement and age. Model systems offer the option of outstanding control over the environment, diet, and genetic background. In theory, it would be possible to track individual movement rates in flies, worms, and mice for thousands of individuals, many more than is practical for human beings. Yet, in general, such studies have not been undertaken.\n\nIn contrast, most activity in geroscience research using popular model systems has focused on increasing lifespan, with the implicit understanding that if one statistically increases lifespan by even a few percent, then one is by definition working on mechanisms germane to the study of aging18. Increased lifespan is often de facto equated with an aging mechanism and is considered the gold standard in geroscience. Yet, for the vast majority of such reports, there is a corresponding lack of knowledge as to whether or not healthspan is increased concomitant with lifespan extension. There are hundreds of publications that identify and characterize genes that “regulate aging”. In contrast, research on defining healthspan (epitomized through studying movement, for example) is relatively unexplored. However, understanding healthspan in these model systems is an absolute prerequisite for beginning to develop pharmacological approaches that extend life in human beings. The reason this is so critical is that increased longevity without increased healthspan is a non-starter. It is unclear whether or not increased lifespan equates to increased healthspan in model systems in general. The prior statement may be considered provocative, as there are many reports in the literature that claim healthspan is increased with lifespan. These studies typically focus on a single gene, which, when mutant, increases lifespan. Such studies, however, typically raise more questions than answers, and these questions need to be robustly addressed before we can unequivocally make the statement that increased healthspan is concordant with increased lifespan for genetic or pharmacological interventions in aging.\n\nIt is encouraging that this area of geroscience is beginning to receive more attention. This is exemplified in C. elegans with the recent publication of two diametrically opposing articles: one group concluded that increased healthspan of the highly cited longevity mutant daf-2 results in decreased healthspan (poorer health with longer life)59. Another group argued the exact opposite (maintenance of health with longer life)60. Both studies have merit, but both studies sampled the available biological space of movement over life with low resolution. For example, in the study by Bansal et al., movement was assessed for just five minutes every fifth day to determine movement rates over lifespan. This sampling represents roughly 0.07% of the potential biological space in the five-day period. As there was some concordance between replicate measures over time, it was assumed that the measured movement rates were consistent throughout the day and night. No data are provided to support this assumption; however, similar to Bansal et al., Hahm et al. also carried out fractional sampling of the biological space in their assessment of movement with age. They collected just five seconds of movement data out of every 24 hours (0.006% of the potential biological space) and claimed this as being representative. In addition, the numbers of animals measured in both studies are quite modest, being of the order of a few dozen individuals measured at most, rather than hundreds or thousands that would be typical in human studies. Both of these studies on aging C. elegans used more objective approaches to quantitate and track movement, and the research community is rapidly moving away from the more subjective measures of the past49,53,58. Both studies also raise a number of intriguing questions with regard to definitively answering whether or not healthspan is increased with lifespan in C. elegans (or Drosophila, or even mice):\n\nWhen measuring movement in a specific time interval, does the amount of movement per time interval change throughout the course of a day/night? What is the impact of circadian rhythms for various genetic or pharmacological interventions?\n\nHow often should one measure movement throughout a lifespan? What is the appropriate measure to determine movement? Many possibilities exist: for example, maximum velocity, total distance moved per unit time, or perhaps a combination of metrics?\n\nDo movement rates change over lifespan with different diets/laboratory environments? What is the impact of variation between labs?\n\nDo movement rates over life change between different strains/species? Is there scaling of healthspan relative to lifespan between strains/species?\n\n\nCross-sectional versus longitudinal study design\n\nMany of the questions posed above can be comprehensively answered using automated video capture systems, and appropriate computational infrastructure, coupled with longitudinal analysis. Longitudinal study design is by many considered to be the gold standard in human trials and permits incorporating within-subject variation as well as between-subject variation. Cross-sectional approaches (young to old, for example) largely miss incorporating such variance. Analysis of healthspan in geroscience should be turning to human clinical trials for guidance on experimental design, and longitudinal analysis has many advantages over cross-sectional experimental approaches61.\n\n\nMaximizing the advantages of model systems in geroscience research\n\nIt seems clear from multiple studies over the last several decades that there is a generalized decline of movement with age in C. elegans and Drosophila. However, we currently do not have sufficient information to subsample a fraction of the animal’s life for movement and then assume that measure is representative over the entire lifespan. C. elegans move with distinct speeds and patterns of movement dependent on the presence of food and their age. It is entirely feasible to thoroughly enumerate this over life. Such data tracking would then allow us to determine how representative a sample of five seconds of movement is for each 24 hours. This kind of rigor should be applied more generally in geroscience experimental design, and the advantages of the experimental system should be exploited, not minimized. Such methodological concerns also apply to genes that have been linked to increased lifespan. For example, if the model organism’s lifespan is increased by 50%, then is it a healthier 50%? Is the lifespan change reflected by increased, sustained, or reduced activity levels? These questions may seem somewhat mundane and not as exciting as mapping pathways or identifying additional genes that modulate lifespan using conventional genetic approaches. However, we currently do not know the answer to most of these “quality of life” questions for many genes or pharmacological interventions, and therefore it makes it very difficult to answer with precision whether or not drug/gene X is improving healthspan. There is a growing effort to acknowledge these issues44,62 and better define healthspan as something that is standardized. More precise experimental definitions of healthspan will allow us to determine clear and unambiguous outcomes that may be translationally relevant, allowing us to capitalize on the strengths of the invertebrate systems.\n\n\nTechnology is a moving target in geroscience\n\nContinuing the discussion of movement as a proxy for healthspan, how should one measure movement in invertebrate model systems of aging? Movement of C. elegans on the two-dimensional surface of the agar plates on which they are typically housed (with or without food) is conceptually simple to track with age. This can be done in either liquid or solid media, although liquid media is not common in aging studies. Liquid media may have additional concerns as an experimental medium, as C. elegans did not evolve in an aquatic environment. There are also newer approaches to measure movement using microfluidic chips63–65. However, such chips may remain somewhat specialized and may not be widely adopted owing to laboratory-specific expertise. Quantitation of movement in Drosophila is more difficult, as adding a third dimension (flight) makes evaluation of the inherent dynamics of movement more problematic. Here too, there have been encouraging efforts using sophisticated cameras/computational approaches to document flight speed and activity with age55–57,66. There are also some more “low-tech” approaches to quantitating Drosophila healthspan with regard to movement (for example, climbing activity67). Such approaches are somewhat more subjective and may suffer from lab to lab variation with regard to implementation. Tinkerhess et al. describe a device in detail for “exercising” Drosophila, which may introduce some standardization in this problematic area. However, whether or not such standards become common practice will depend on the degree of adoption by the greater research community. Widespread adoption of a commonly agreed upon method for evaluating movement is critical for replication purposes. Having focused on movement as being the gold standard for healthspan measures in aging invertebrates, there are some alternative measures that have also been employed to assess healthspan, but these tend to be more idiosyncratic and may be model specific, so that the translational relevance to human aging is not clear.\n\n\nOther healthspan metrics in invertebrate models\n\nAlthough a decline in cell number/cell volume for multiple tissues has been documented in aging human beings for several tissues, similar approaches in model systems in aging are not as well established. Adult C. elegans comprise 959 cells across multiple tissues, including the musculature, nervous system, pharynx, intestine, reproductive organs, and epidermis. Perhaps the closest parallel of tissue-specific aging in worms compared to humans is the loss of muscle mass with age. Loss of muscle mass is well established in human beings and is termed sarcopenia68. Recently, the van Loon group concluded that the loss of muscle mass with age can be explained by atrophy of type II fibers and the commonly held belief that individual fiber loss with age was erroneous69. What makes this particular study so compelling is that it was done on the same individuals over time, in contrast to previous studies which were largely cross-sectional in nature (i.e. young versus old). The definition of sarcopenia is constantly being re-evaluated and is currently defined not only by loss of muscle mass but also by loss of muscle quality (i.e. weakness)70. Loss of muscle mass in aging worms was first observed by the Driscoll group in 2002 in a seminal paper describing various aspects of the pathobiology of the aging worm53. It was reported that the 95 individual cells comprising body wall muscle were observed to atrophy and fragment with age, visualized through muscle-specific green fluorescent protein (GFP) reporters53. On the surface, it would appear to be difficult to measure muscle quality (strength) in worms, but recent advances using microfluidic technology have enabled force measurements to be evaluated for worms captured in a microfluidic device. Young worms exert ~34 µN of force when thrashing in liquid media and can move specialized posts in a microfluidic device a distance of 20.36 µm63. This type of methodology could be applied to aging worms in conjunction with muscle-specific reporters as in Herndon et al. to evaluate not only muscle quantity with age but how well the muscle functions. Arguments for other potentially related measures such as thrashing rate in different density liquids can also be made, but it is far from clear how such measures relate to sarcopenia in mammals.\n\nMuscle is not the only tissue to degenerate in aging worms. We previously evaluated intestinal integrity with age and determined that there was a stochastic degradation as well as a decrease in the absolute number of cells comprising the intestine71. Presumably, this change has functional consequences for the digestion of food in aging animals. However, it is difficult to relate such outcomes to intestinal aging in mammals, as there is no clear homologous pathology in the elderly. We also reported a loss of specific hypodermal nuclei with age in C. elegans72, but, again, the implications for the healthspan of the aging worm are not straightforward. One of the more striking features of the pathobiology of the aging worm is a substantial growth of uterine masses with age72,73. This seems to be a robust phenomenon of nematode aging having been qualitatively described in a previous report74. This germline pathology appears to be modulated by a decline in cep-1/p53 with age73. One clear outcome of the increase in uterine masses in the aging nematode is the massive proliferation of DNA copy number per worm. As individual animals age, there is as much as a fivefold increase in genome copy number per worm, directly related to endoreduplication in the gonad. The implications for the health of the animal are again not clear, and it is even less clear if there is a straightforward parallel to healthspan in aging humans.\n\nThe widely used long-lived mutant daf-2(e1370) has nearly double the genome copy number per individual animal compared to the wild-type73. This is observed even in young animals with the daf-2(e1370) allele, despite being somewhat less fertile than wild-type controls and containing less progeny. It is formally possible (but unlikely) that the extra genome copies are due to additional somatic cells indirectly derived from the daf-2 mutation. Alternatively, perhaps there is endoreduplication of specific cell types. Unfortunately, the origin of these extra genome copies currently remains unknown. More work is needed with regard to genome/cell number in the aging worm. One of the worm’s clear strengths is that it remains almost unique in experimental systems in that a complete understanding of the cell fate map from development to adulthood has been elucidated. It is possible that extra genomes in the daf-2 mutant allow for an increased reserve capacity against somatic mutations with age and therefore maintenance of tissue homeostasis. Such an explanation has been advocated to explain the resistance of elephants to cancer, as they have 20 copies of the tumor suppressor gene p53, as opposed to humans, who have only one. On a cell number basis alone, elephants would be expected to have much more cancer late in life than ourselves, yet they have a cancer incidence of only 4.8% compared to 11–25% in ourselves75, perhaps due to the extra copies of p53 in the elephant genome allowing for more robust tumor suppression. Similarly, perhaps critical extra genome copies in the daf-2 background provide a “reserve capacity” buffering life-limiting pathologies in aging worms. Regardless, the increased genome copy number in daf-2 is at present a curiosity, and the functional consequences remain unexplained.\n\nOther pathological hallmarks that appear to change with age in C. elegans include altered neuronal architecture of aging worms76,77 and an increase in age-related pigments78. There have also been reports of a decline in reproductive fitness with age in C. elegans79. Reproductive health is generally not a focus of geroscience, as the elderly face many more serious health problems than their ability to reproduce. For a number of the diverse aging phenotypes reported in C. elegans, many seem to arise well before mean lifespan, and the dynamics over life from lab to lab or influence of genetic background are typically not known. In Drosophila too, there have been a number of reports of age-related changes in different organ systems such as the intestine and germline13. Again, the functional consequences for healthspan are not clear for reasons similar to those articulated in describing the aging worm intestine. For a tissue-specific decline in organ function with age, the fly has one clear homologue of human organ aging: it has a beating heart with many features in common with the mammalian heart and has been used to investigate invertebrate cardiac aging in a number of studies14. Remarkably, there have even been reports describing the benefits of exercise on the aging fly heart67,80, and this is an exciting research area which needs to be more broadly studied. Unfortunately, there are only a few labs that have the ability to assess cardiac function in the context of diet, genetic background, or individual variation. Given the plethora of genetic tools and strains available in Drosophila, a more widespread investigation of cardiac aging would be very powerful to help address functional changes in the aging Drosophila heart. Regardless of the reported association with age of each of these diverse phenotypes, they are often reported in the context of healthspan. However, without understanding the functional consequences for the aging animal with a high degree of precision, it is difficult to relate such measures to homologous outcomes of healthspan in human beings.\n\n\nHealthspan measures in aging mice\n\nFunctional decline in human beings occurs with increasing age, including a decrease in activity, cognition, bone quality, and other multiple reduced organ or tissue functions. We know that such systems decline from endogenous mechanisms of aging, as the performance of elite athletes of all disciplines declines with age quite markedly. One can make the argument that human physiology is optimally defined in an elite athlete, in which diet, lifestyle, and environment have all been optimized to produce peak performance. Yet, even in these individuals, each functional domain of aging declines with age. However, for mice, much of the data describing similar functions are relatively poorly characterized, relying on data from a few recent studies81,82 or reports from several decades ago. Data on healthspan in mice generated from the 1990’s and before are particularly difficult to relate to contemporary studies. This is because of animal housing practices being quite different in the past compared to current standards of care. In stark contrast to our understanding of healthspan with age in human beings, we know remarkably little about the impact of diet, housing, and genetic background on functional domains of healthspan in mice. Much work needs to be done to address this deficit before we can begin to reasonably assess whether or not pharmacological interdiction with any intervention in aged mice slows or improves function in specific tissues62. Particularly exciting is the development of new technologies that enable non-invasive surveillance of many critical tissues in live mice. Many of these technologies did not exist prior to the turn of the century, so there are exciting opportunities to define in exquisite detail functional decline in different tissues and systems in multiple genetic backgrounds and species83–85. For example, amazing advances in cardiovascular surveillance via ultrasound with fantastically high frame rates (>1000 frames/second) are possible, facilitating the study of vessel aging in vivo86. Improvements in micro-computed tomography (micro-CT) enable whole body scans in as little as eight seconds with minimal radiation exposure at excellent resolution to allow the study of in vivo bone aging (http://bruker-microct.com/products/1278.htm). Whole-body metabolism and activity can also be studied over time with extremely high data rates (data collected every second for days!) with new advances in metabolic cages (http://www.sablesys.com/products/promethion-line/promethion-cages/). There are also tremendous advances in the assessment of function in the brain via positron emission tomography/single photon emission CT (PET/SPECT) and magnetic resonance imaging (MRI), with extraordinary detail being revealed through these powerful new imaging technologies. Suffice to say that all these improvements in longitudinal surveillance of aging animals provides enormous opportunity to define in great detail how tissues change in function with age in conjunction with targeted pharmacological interventions.\n\n\nPharmacological intervention for increased healthspan/lifespan\n\nSince the early 2000’s, there has been an increasing focus in the study of aging by manipulating lifespan through pharmacological approaches20,22,23,29,59–62,87–90. The 1990’s could be argued to be the era of “genes for aging” in geroscience research, and in the second decade of the 21st century, there has been an explosion of interest in identifying robust pharmacological interventions for lifespan. Healthspan effects have been a secondary consideration until now, but this too is changing with increasing reports of late-life interventions in aging mice to increase lifespan, coupled with healthspan studies28,91–96. The intervention testing program administered by the National Institute on Aging (NIA) has been an invaluable advocate in developing this concept97. Initially formulated in the early 2000’s as a multi-center testing vehicle for “pro-longevity” agents, it has popularized the experimental design of a multi-site trial for intervening in aging. The Intervention Testing Program (ITP) consists of three geographically distinct sites (University of Michigan, University of Texas Health Sciences Center, and Jackson Labs), each of which independently evaluates the efficacy of specific pharmacological interventions for extending lifespan in a single strain of genetically diverse mice. The goal of the ITP is to robustly identify interventions that extend life, and although interventions are tested from young adults in some cases, the main goal is to identify late-life interventions. This approach is especially relevant when one considers translational impact, as it is difficult to imagine prescribing a pro-longevity intervention to young adult humans. Far more realistic are targeted efforts in the elderly population. More recently, the ITP has begun to transition from evaluating lifespan alone to assessing select functional outcomes. This is a welcome development, although functional outcomes need to be carefully characterized in the context of human aging if the maximal impact is to be realized. Detailed investigations into the variance of aging phenotypes in untreated animals with functional consequence are a necessary pre-requisite in the effort to precisely understand the impact of any potential pharmacological interdiction.\n\nThe overall ITP approach has also given birth to the Caenorhabditis ITP (CITP) program. The goals of the CITP are very similar to that of the ITP, but it focuses on identifying robust chemical responses across distinct genetic backgrounds by utilizing genetically diverse species and strains of nematodes. The CITP too has three geographical testing sites for the purposes of replication: the Buck Institute for Research on Aging, Rutgers University, and the University of Oregon. The CITP program is attempting to standardize many aspects of geroscience (survival, lifespan extension, etc.) in the aging worm and to assess healthspan as well. One can see a future in which interventions are evaluated in the CITP program and chemical “hits” that robustly affect lifespan at all three sites are then evaluated for healthspan (movement is perhaps the low-hanging fruit here). Such hits would then subsequently be prioritized for testing in the ITP. The ITP today has evaluated at least 25 interventions in mice and has an approximately 10% hit rate in terms of statistically significantly increasing lifespan. It is beyond the scope of this article to discuss in detail the many pharmacological approaches reported for intervening in aging. However, it is worth discussing two highly visible examples in this area.\n\nIf interventions that are robustly positive for lifespan extension are also positive for healthspan extension, then we have a very powerful system for the prioritization of preclinical interventions for aging in human beings. Arguably, rapamycin is the first robust outcome from the ITP in this regard, with multiple reports of lifespan extension in mice and some reports of healthspan extension as well (Table 1). We previously reported that cardiac health in elderly female mice was improved by a short rapamycin treatment late in life26. This was later confirmed in similar experiments by another group27. However, in another investigation on late-life rapamycin treatment in males only, no significant benefits were reported25. In addition, there are clear deleterious effects from chronic rapamycin treatment in mice. Negative outcomes include testicular atrophy and increased incidence of cataracts28. Clearly, more work needs to be done to address the potential for sex-specific responses to rapamycin with regard to healthspan effects as well as adverse consequences resulting from pharmacologically attenuating aging. We are clearly in the beginning of developing and characterizing robust interventions in preclinical models for aging, but where are we in human trials?\n\nPreliminary trials in human beings to reduce morbidity and extend healthspan/lifespan are either in process or in the planning stage at multiple sites around the world. These efforts are in part capitalizing on the outcomes from geroscience in model organisms over the last three decades. One example is the TAME (Targeting Aging with MEtformin) trial, recently discussed in the popular press and literature98. This trial is built in part on successful studies in aging model systems treated with metformin20,99,100 as well as data from a recent meta-analysis of diabetics. A significant motivating factor in this trial is the excellent safety profile of metformin, which has been in use for nearly 60 years. The approach is to determine whether chronic metformin treatment in the elderly improves health and reduces co-morbidity for multiple indications. Other work recently completed in this context is a limited trial with the mTOR inhibitor RAD001—a molecule similar to rapamycin that also decreases mTOR activity101. This trial focused on a vaccine response in the elderly: older individuals were pre-treated with RAD001, which, perhaps counterintuitive to conventional wisdom, resulted in an improved immune response to an influenza vaccination compared to an untreated control group. This is consistent with a variety of model systems in a geroscience context in that down regulation of mTOR appears to benefit function for many systems (including the immune system) in aged animals. One commonality in both candidate interventions is the fact that the interventions were already FDA approved and have known safety profiles. This type of approach is likely to be the most straightforward way to aggressively move into trials for intervening in aging, as the length of time required to develop novel pharmacological interventions will require many years and is subject to stringent approvals at multiple levels.\n\nRegardless of the initial success or failure of initial candidate molecules in the human arena, it is quite likely that the pace of such work will increase in the near future owing to growing demand for biomedical solutions to increasing healthcare costs as the baby boomer generation continues to age. The conserved biology of aging coupled with multiple successes in extending lifespan/healthspan in geroscience research on model organisms give a great deal of hope that we will identify effective and precise therapeutics to combat the functional decline of aging and perhaps increase lifespan as well.",
"appendix": "Competing interests\n\n\n\nThe author declares that he has no competing interests.\n\n\nGrant information\n\nSimon Melov was supported in part by NIH AG045844 and AG051129.\n\n\nAcknowledgements\n\nThanks to the reviewers and Gordon Lithgow for helpful comments.\n\n\nReferences\n\nJohnson TE: Increased life-span of age-1 mutants in Caenorhabditis elegans and lower Gompertz rate of aging. Science. 1990; 249(4971): 908–12. PubMed Abstract | Publisher Full Text\n\nFriedman DB, Johnson TE: Three mutants that extend both mean and maximum life span of the nematode, Caenorhabditis elegans, define the age-1 gene. J Gerontol. 1988; 43(4): B102–9. PubMed Abstract | Publisher Full Text\n\nJohnson TE, Wood WB: Genetic analysis of life-span in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1982; 79(21): 6603–7. 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}
|
[
{
"id": "13660",
"date": "29 Apr 2016",
"name": "Monica Driscoll",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13661",
"date": "29 Apr 2016",
"name": "Nathan LeBrasseur",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-785
|
https://f1000research.com/articles/5-783/v1
|
29 Apr 16
|
{
"type": "Review",
"title": "Rho, ROCK and actomyosin contractility in metastasis as drug targets",
"authors": [
"Irene Rodriguez-Hernandez",
"Gaia Cantelli",
"Fanshawe Bruce",
"Victoria Sanz-Moreno",
"Irene Rodriguez-Hernandez",
"Gaia Cantelli",
"Fanshawe Bruce"
],
"abstract": "Metastasis is the spread of cancer cells around the body and the cause of the majority of cancer deaths. Metastasis is a very complex process in which cancer cells need to dramatically modify their cytoskeleton and cope with different environments to successfully colonize a secondary organ. In this review, we discuss recent findings pointing at Rho-ROCK or actomyosin force (or both) as major drivers of many of the steps required for metastatic success. We propose that these are important drug targets that need to be considered in the clinic to palliate metastatic disease.",
"keywords": [
"Rho-ROCK",
"actomyosin",
"metastasis",
"Rho"
],
"content": "Introduction\n\nMetastatic disease is still largely incurable because of its systemic distribution and resistance to current therapies, and it is the cause of more than 90% of cancer-related deaths1,2. In spite of its clinical importance, the underlying cellular and molecular mechanisms of cancer metastasis are only partially understood3. Thus, improved knowledge of how cancer cells acquire metastatic traits is necessary to unravel novel drug targets and prognostic markers of distant relapse.\n\nMetastasis is a complex multi-stage process by which cancer cells disseminate from primary tumors, survive in distant sites and eventually grow as secondary tumors3. The main events of the metastatic cascade involve loss of cell-cell contacts, cancer cell migration, local invasion of the surrounding extracellular matrix (ECM), interactions with stroma, intravasation and transit into blood or lymphatic vessels, arrest at secondary sites, extravasation, survival and colonization of distant sites4. Genetic alterations and deregulation of critical oncogenic signaling pathways affecting survival, proliferation, apoptosis, and cell motility, regulate many of these complex metastatic events3,5. In addition, the interaction with the tumor microenvironment such as ECM, growth-supportive stromal cells, inflammatory cells and endothelial cells strongly impacts the metastatic capabilities of cancer cells6,7.\n\nMany signaling pathways have been reported to have an impact on metastasis and have been the focus of excellent reviews8–15. In the present review, we will focus on Rho-ROCK signaling and actomyosin contractility, key regulators of several main steps in metastasis. Rho-ROCK, through its actions on cytoskeletal dynamics and through regulation of critical signaling pathways, controls several cellular processes important for metastasis such as cell migration, local invasion, survival at the secondary site, and tumor outgrowth16–18.\n\n\nRho GTPases and metastasis\n\nThe Rho family of small GTPases plays crucial roles in the regulation of the actin cytoskeleton, cell polarity, cell migration, cell proliferation, invasion, and metastasis19. Rho GTPases act as molecular switches cycling between a guanosine triphosphate (GTP)-bound active state and guanosine diphosphate (GDP)-bound inactive state to translate extracellular signals into different cellular responses19. Their activity is controlled by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs)18. The best studied and most conserved Rho family members across eukaryotic species are Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division control protein 42 homolog (Cdc42), and Ras homolog gene family member A (RhoA)18. Rac1 stimulates lamellipodia formation20, whereas RhoA regulates the formation of stress fibers or favors amoeboid migration depending on the cellular context and the properties of the matrix. RhoA bound to GTP leads to activation of its effectors Rho-associated protein kinases (ROCK1 and ROCK2)21–23. ROCK1/2 serine/threonine kinases promote actomyosin contractile force generation by decreasing myosin phosphatase activity and thereby increasing phosphorylation of myosin light chain 2 (MLC2)24. On the other hand, Cdc42 induces filopodia formation25, but Cdc42 signaling can also generate actomyosin contraction through p21 protein (Cdc42/Rac)-activated kinase 2 (PAK2) and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) kinases26,27. Deregulation of the Rho-ROCK signaling pathway has been found in a variety of cancer types and in several cases correlates with disease progression28–30 (Table 1). Furthermore, inhibition of ROCK signaling could suppress migration and invasion in vitro and impair the metastatic process in vivo, suggesting that ROCK inhibitors might be potential anti-metastatic agents30–32.\n\nShown are examples in the literature of where different stages of the metastatic cascade have been shown to be influenced by Rho-ROCK and/or actomyosin contractility signalling. (SCC= Squamous cell carcinoma)\n\n\nRho/ROCK signaling and actomyosin contractility in early dissemination\n\nThe ability of cancer cells to migrate into and invade surrounding tissue is a critical step in the metastatic cascade, which requires increased cell motility driven by altered cytoskeletal organization and contacts with the ECM and the stroma33. Cancer cells can move either collectively or as individual cells34,35. The majority of tumors originate from epithelial tissues, and epithelial cancer cells that leave the primary tumor undergo a complex program called epithelial-mesenchymal transition (EMT). Incomplete or partial EMT allows collective migration in which cells can maintain cell-cell adhesions and migrate collectively in a coordinated manner as strands, sheets, or cell clusters. On the other hand, complete EMT is associated with the loss of cell-cell adhesions in favor of cell-ECM interactions and the concomitant acquisition of individual migratory characteristics36,37. After undergoing EMT, individual cancer cells can engage into elongated mesenchymal or rounded amoeboid modes of movement, distinguished by their different usage of signaling pathways. Mixed mesenchymal and amoeboid phenotypes have also been identified38,39. Individual cell migration seems to be required for blood-borne metastasis40.\n\nActomyosin contractility driven by Rho or ROCK signaling is key in controlling tumor dissemination, as all forms of cell migration require a certain degree of actomyosin force34,41. During collective cell migration, actomyosin contractility is high around the edges of groups of invading cancer cells, which generates pulling forces between the substrate and the follower cells, together with a prominent actomyosin ring at lateral regions of the groups to maintain coupling between cells and collective forward movement42,43. On the other hand, in individual migration, the contractile cortex is crucially important for amoeboid to intermediate forms of movement, and some degree of contractility is also required to retract protrusions in mesenchymal migration39,44–46. The mesenchymal mode of movement is characterized by an elongated, spindle-like shape, high levels of adhesion, and Rac-dependent adhesive actin-rich protrusions23,46,47. On the other side of the spectrum, in amoeboid migration, cancer cells adopt a rounded or irregular morphology with blebs as functional protrusions. Amoeboid motility is promoted by high levels of RhoA/Ras homolog gene family member C (RhoC) or ROCK-driven actomyosin contractility and requires lower levels of adhesion that allow higher speeds of movement46–50.\n\nCancer cell migration is a dynamic process, and individual cancer cells can switch between modes of movement to adapt to the changing microenvironment and facilitate tumor dissemination. Different cues will favor either a mesenchymal-amoeboid transition (MAT) or an amoeboid-mesenchymal transition (AMT)23,45,49,51,52. Their core regulatory network is the mutually inhibitory circuit between Rac1 and Rho GTPase signaling in migrating cells (Figure 1). Higher Rac1 activity promotes cell elongation and permits long actin-rich protrusions characteristic of mesenchymal migration. Moreover, active Rac1 negatively regulates Rho or ROCK signaling and suppresses amoeboid movement. On the other hand, active Rho or ROCK supports bleb-based amoeboid migration23,45,49,51,52 and limits excessive Rac1-dependent adhesion via regulation of the Rac GAPs ARHGAP22 and filamin-A-associated Rho GTPase activation protein (FilGAP)23,53. Furthermore, cancer cells control amoeboid migration at the transcriptional level under circumstances in which matrix compliance allows sustained actomyosin contractility (Figure 1). Different chemical cues have been shown to control this process. For instance, amoeboid melanoma cells support contractility, establishing a positive feedback loop with the cytokines leukemia inhibitory factor (LIF)/IL6 and the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway to maintain Rho-ROCK activity49. As a result of high STAT3 activity, very contractile cells secrete different factors, including matrix metalloprotease 9 (MMP-9). MMP-9 promotes the generation of actomyosin contractile force and bleb-driven migration through a positive feedback loop via CD44 binding and increased MLC2 phosphorylation to sustain amoeboid invasion48. Moreover, amoeboid contractile cells secrete high levels of transforming growth factor beta (TGFβ), and downstream of it a Sma- and Mad-related protein 2 (SMAD2)-Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain (CITED1) transcriptional network sustains actomyosin contractility54. In addition, the physical properties of the matrix play an important role in establishing a balance between actomyosin levels and adhesion to regulate optimal migration efficiency34,39,47,55,56. Increased ECM density results in increased matrix stiffness, in which cells sense and respond by increasing Rho-mediated actomyosin contractility57. Furthermore, slow mesenchymal cells can switch to fast amoeboid migrating modes under conditions of low adhesiveness and high physical confinement47,56.\n\nROCK-driven actomyosin contractility is stimulated by extracellular signals such as leukemia inhibitory factor (LIF) and transforming growth factor beta (TGFβ) to promote rounded amoeboid cancer cell motility. Rounded amoeboid cells display blebbing as well as high levels of actomyosin contractility and a rounded morphology. They interact with the extracellular matrix (ECM) by physically deforming it and by secreting metalloproteases (MMPs). In the stroma, ROCK-driven actomyosin contractility promotes the transformation of fibroblasts into cancer-associated fibroblasts (CAFs), driven by Yes-associated protein (YAP) as well as by extracellular factors. Blue indicates positive regulators of contractility, purple indicates negative regulators of contractility and orange lines indicate actomyosin contractility. Abbreviations: CAF, carcinoma-associated fibroblasts; CITED1, Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain, 1; FilGAP, filamin-A-associated Rho GTPase activation protein; JAK, Janus kinase; RhoA, Ras homolog gene family member A; ROCK, Rho-associated protein kinase; SMAD2, Sma- and Mad-related protein 2; STAT3, signal transducer and activator of transcription 3.\n\nThe ability to switch between different modes of migration is an important factor for metastatic dissemination, as cancer cells have to migrate through a range of ECMs to escape the primary tumor and spread to distant organs. Therefore, anti-metastatic treatments should target the ability of tumor cells to cope with such variability. Recently, it has been described that potent ROCK inhibitors are able to strongly inhibit actomyosin contractility and collapse the actomyosin cytoskeleton, blocking both mesenchymal and amoeboid modes of movement32.\n\nIntra-vital imaging studies have shown that bleb-driven highly contractile amoeboid migration is favored in the invasive fronts of melanomas and breast cancers23,29,45,48,49,58. Furthermore, in these studies, it has been shown that treatment with ROCK inhibitors or actomyosin perturbations (or both) is able to decrease tumor cell motility in vivo23,29,32,45,49,58. Hence, ROCK inhibition could effectively impair local invasion and dissemination of cancer cells (Figure 1).\n\nWithin the tumor, a variety of non-cancer stromal cells interact with the cancer cells promoting tumorigenesis7. Actomyosin contractility not only is fundamental for cancer cell migration and invasion but also is crucial for maintenance of the carcinoma-associated fibroblasts (CAFs) phenotype, an important stromal component in the tumor microenvironment7. Actomyosin contractility activated by ROCK signaling and the LIF/JAK/STAT pathway is crucial for CAF-dependent pro-invasive physical remodeling of the ECM favoring tumor aggressiveness and dissemination42,49,59,60. Additionally, actomyosin contractility, Src function, and matrix stiffening induced by TGFβ, are required for Yes-associated protein (YAP) activation in CAFs to promote ECM remodeling and cancer cell invasion, and to generate a positive feedback loop that helps to maintain the CAF phenotype61 (Figure 1). Moreover, contractility in CAFs has been shown to modulate EMT and metastasis-initiating cell properties in breast cancer models62.\n\nTherefore, some degree of actomyosin contractility is essential for both cancer cells and stroma for efficient cell movement in the initial steps of the metastatic cascade34,41,49,59,61, and some factors such as TGFβ and LIF can stimulate contractility both in cancer cells and in fibroblasts.\n\n\nRho/ROCK signaling and actomyosin contractility in transendothelial migration\n\nAfter local invasion within the primary tumor microenvironment, cancer cells need to spread throughout the body and colonize new organs to form metastases. They do so by exploiting the vascular and lymphatic systems. The process through which cancer cells enter and exit vessels crossing the endothelial layer is known as transendothelial migration, which is extremely complex and involves the interaction with several different cell types, such as platelets, immune cells and endothelial cells, and the activation of a variety of signaling pathways63. These events are in some cases similar to those occurring during inflammation or infection, when immune cells need to enter and exit vessels. In fact, parallels between cancer cell and immune cell migration allow for interesting speculation in areas of cancer cell dissemination that are still not fully understood.\n\nIntravasation. The first step in this metastatic cascade is intravasation, the entry of tumor cells into blood vessels. Intravasation depends on the weakening of cell-cell junctions between endothelial cells, which allows cancer cells to squeeze in between adjacent endothelial cells and enter the vessel lumen63. From a molecular perspective, not as much is known about intravasation compared with other steps in the metastatic cascade as this is an experimentally challenging step to study64,65. In fact, intravasation is dependent on the ability of cancer cells to invade towards blood vessels, so it is difficult to distinguish between genes involved in invasion and intravasation63. RhoA signaling has been linked to the process of intravasation66 (Figure 2). Specifically, RhoA activity in cancer cells is thought to be stimulated by macrophage contact and leads to the formation of invadopodia. Invadopodia are instrumental in the degradation and eventual breakdown of the matrix barrier, which allows for tumor cell intravasation. Furthermore, highly contractile, rounded amoeboid melanoma cells have been shown to intravasate more efficiently than low-contractility elongated cells in vivo67,68. Once in the bloodstream, cells are transported throughout the body by the blood flow (Figure 2).\n\nRhoC/ROCK signaling promotes survival of cancer cells in the blood flow as well as adhesion to the endothelium and extravasation. ROCK-driven actomyosin contractility within endothelial cells can be stimulated by secreted factors and is essential for cancer cell extravasation. Abbreviations: RhoC, Ras homolog gene family member C; ROCK, Rho-associated protein kinase.\n\nExtravasation. Eventually, cancer cells flowing through the bloodstream need to exit blood vessels to form secondary tumors. This process is known as extravasation and entails several sequential steps. First of all, cancer cells form loose adhesions to the vascular endothelium, which is known as tethering. These loose adhesions then are tightened to form firm adhesions: firmly adhering cells then can cross the endothelial barrier and extravasate63.\n\nThe best-studied mechanism for extravasation is known as paracellular extravasation, during which cancer cells exit the vessel by squeezing in between endothelial cells. An alternative mechanism for cancer cell extravasation is transcellular extravasation, where tumor cells exit the vessel by going through endothelial cells63,69. Transcellular extravasation has been observed in immune cells70 and has also been identified in cancer cells, where it probably plays a role in some cases71.\n\nRho or ROCK-driven actomyosin contractility within extravasating cells has been shown to play an important role. For instance, in monocytes, RhoA activity has been shown to be necessary for tail retraction during extravasation72. In the context of transcellular extravasation, monocytes can rely on RhoA and ROCK signaling73,74.\n\nOn the other hand, in prostate cancer cells, it is RhoC and ROCK signaling that is essential for interaction with endothelial cells, promoting adhesion and paracellular extravasation75. As a result of its role in promoting extravasation, RhoC signaling is a key driver of tumor dissemination and metastasis75, in part explaining how RhoC was one of the first genes identified as a metastasis driver76. Furthermore, RhoA and RhoC have been shown to drive adhesion to the endothelium and transendothelial migration in breast and prostate cancer cells77,78. Consequently, rounded-amoeboid cancer cells with high levels of RhoA or ROCK-driven actomyosin contractility are more efficient during transendothelial migration than elongated cells both in vitro and in vivo67,68,79. Additional evidence supporting the importance of RhoA-driven contractility in transendothelial migration comes from studies examining the role of RhoA regulators. For instance, FilGAP, a Rac GAP, promotes RhoA signaling and rounded-amoeboid motility by suppressing Rac, and as a consequence it enhances in vivo extravasation of breast cancer cells53. Conversely, the RhoA GAP ARHGAP7 has been shown to be a negative regulator of transendothelial migration in thymic lymphoma80.\n\nCancer cells that successfully extravasate need to cross the vascular basement membrane that surrounds the vessel63. Since actomyosin contractility has been shown to promote the secretion of proteases in rounded amoeboid cells48, it is tempting to speculate that highly contractile extravasating cells could have an advantage when crossing the vascular basement membrane.\n\nIn order for paracellular extravasation to occur, cancer cells need to weaken cell-cell junctions within the endothelium. This can be mediated by regulating Rho or ROCK signaling and actomyosin contractility within the endothelial cells themselves (Figure 2). Lung cancer cells have been shown to induce adherens junction disassembly by stimulating actomyosin contractility through Rho/ROCK in endothelial cells81. Furthermore, thrombin stimulation of endothelial cells has been shown to induce ROCK activity and subsequently lead to cytoskeletal remodeling, junction disruption, and endothelial permeability82,83. Tumor-derived thrombin induces endothelial gap formation and transendothelial migration84. Furthermore, cancer cells have been shown to use thrombin within blood vessels in order to promote metastasis85. This prompts the speculation that actomyosin contraction in endothelial cells could be controlled by thrombin produced by cancer cells.\n\nAs well as leading to junction disassembly, actomyosin contractility in endothelial cells allows for endothelial cell retraction86,87, which increases endothelial permeability. Moreover, ROCK-driven actomyosin contractility in endothelial cells has been shown to prevent endothelial cell re-spreading downstream of ephrin-B signaling, which maintains increased endothelial permeability88. Conversely, ROCK inhibition has been shown to decrease endothelial permeability after hemorrhage89,90. Although these studies have not been conducted in cancer models, ROCK activity in endothelial cells could be similarly regulated while in contact with disseminating cancer cells.\n\nIn brief, we speculate that the ability of cancer cells to form secondary tumors is to a certain extent dependent on their ability to manipulate the cytoskeleton of endothelial cells; thus, increasing endothelial permeability could be a crucial step to promote extravasation. More work is needed to validate the roles of Rho/ROCK or actomyosin contractility (or both) in tumor cells during both cancer intravasation and extravasation.\n\n\nRho/ROCK signaling and actomyosin contractility in metastatic colonization\n\nFollowing extravasation at secondary sites, cancer cells that survive can form micro-metastasis and colonize new sites. In order for this colonization to take place, cancer cells must be able to adhere to endothelial cells, extravasate, survive and proliferate at the secondary site. The first few hours of colonization are crucial in determining the success of this process, as cells will undergo apoptosis if they do not adhere to their new niche. Furthermore, once established, cells must be able to evade the immune response in order to survive91. Although we have discussed that Rho/ROCK signaling is important for early dissemination, there is also evidence to suggest that Rho/ROCK signaling, actomyosin contractility or its regulators, or a combination of these are important for efficient colonization at secondary sites.\n\nIn vivo studies where cancer cells are injected intravenously (i.e., experimental metastasis assays) show that high levels of actomyosin contractility play a role in seeding of and colonizing the lung. For instance, cells selected for efficient colonization in the lung such as the highly metastatic A375M2 melanoma cell line have higher levels of RhoC76, RhoA23 and phosphorylated MLC248 when compared with low metastatic A375P melanoma cells.\n\nSeveral studies have confirmed the importance of the initial hours in seeding during colonization. For example, serum response factor (SRF) co-activators myocardin-related transcription factors (MRTFs) are able to control the expression of MLC292 (Figure 3). MRTF and SRF are both important for early stages of lung colonization in breast cancer and melanoma92. Furthermore, depletion of MLC2 itself has also been shown to reduce lung colonization92. Conversely, enhanced actomyosin contractility favors colonization: for example, depletion of the actomyosin contractility suppressors Rac1 and its GEF dedicator of cytokinesis 3 (DOCK3) favors early lung colonization23. In melanoma, pigment epithelium-derived factor (PEDF) reduces lung colonization and suppresses lung tumor outgrowth93,94. PEDF is a negative regulator of Rho-ROCK signaling through supporting DOCK3-Rac1 activity95 (Figure 3). Furthermore, oncogenic BRAF suppresses phosphodiesterase 5A (PDE5A), which in turn inhibits actomyosin contractility96 (Figure 3). Therefore, re-expression of PDE5A reduces the ability of melanoma cells to colonize the lung and prevents short-term survival and long-term cancer growth in the lung96.\n\nActomyosin contractility promotes cancer cell colonization and outgrowth at a secondary site to form metastases. Contractility is under the control of a wide variety of pathways, including SRF/MRTF, TGFβ-SMAD-CITED1, MMP-9, BRAF-V600E and Cdc42 signaling. Blue indicates positive regulators of contractility, and purple indicates negative regulators of contractility. Abbreviations: CITED1, Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain, 1; Cdc42, cell division control protein 42 homolog; MLC2, myosin light chain 2; MMP, matrix metallopeptidase; MRTF, myocardin-related transcription factors; PDE5A, phosphodiesterase 5A; PEDF, pigment epithelium-derived factor; RasGRF2, Ras protein-specific guanine nucleotide-releasing factor 2; ROCK, Rho-associated protein kinase; SMAD, Sma- and Mad-related protein; SRF, serum response factor; TGFβ, transforming growth factor beta.\n\nAs mentioned earlier, Cdc42 can also promote actomyosin contractility in cancer cells26. Further evidence of the importance of actomyosin contractility in early colonization has been shown by experiments in which loss of Ras protein-specific guanine nucleotide-releasing factor 2 (RasGRF2), an inhibitor of Cdc4297, enhanced colonization of the lungs in a Rac-independent manner. This was associated with higher actomyosin contractility levels97 (Figure 3).\n\nTGFβ signaling plays an important role in promoting cancer cell colonization40,54,98 (Figure 3). We recently found that TGFβ increases actomyosin contractility in melanoma cells54. While TGFβ is known to promote EMT99 in epithelial cancers, in melanoma TGFβ signals through SMAD2 and the adaptor CITED1 to support contractile amoeboid migration54. TGFβ no longer sustains lung colonization in melanoma cells if the SMAD2-CITED1 axis is not functional54, which serves to highlight the multiple levels in which actomyosin contractility promotes colonization.\n\nFurthermore, ROCK regulates expression of several MMPs, including MMP-9, which promote early stages of lung colonization48 (Figure 3). While MMPs exert their catalytic function in degradation of the ECM during local invasion, the non-catalytic roles of MMP-9 could promote the survival of cancer cells at the metastatic secondary sites. For example, it has been shown that non-catalytic functions of MMP-9 regulate STAT3 functions to drive survival in B-cell chronic lymphocytic leukemia (B-CLL) cells100.\n\nFrom these results, it is clear that positive and negative regulators of Rho/ROCK signaling or actomyosin contractility (or both) are critical for cancer cells to efficiently colonize the metastatic sites in experimental metastasis models.\n\nWe have highlighted the crucial role that Rho/ROCK signaling or actomyosin contractility play in dissemination and metastatic colonization using a range of experimental cancer models. A highly contractile phenotype is clearly critical for effective cancer colonization, ultimately supporting the idea of developing drugs to inhibit actomyosin contractility. In vivo validation of the role of Rho/ROCK signaling or actomyosin contractility (or both) in metastasis is important to qualify these signaling modules as potential drug targets. Experimental metastasis models are insightful for understanding the processes of extravasation and colonization to the lungs, but recapitulation of the entire metastatic cascade, including local invasion, dissemination and intravasation, requires the use of spontaneous metastasis models101. Indeed, it has recently been shown that a new class of ROCK inhibitors has the ability to prevent both experimental and spontaneous metastases formation32. It will be of great importance to combine these mouse models with non-invasive cell-tracking techniques102,103 to understand the entire process and how early Rho/ROCK signaling should be targeted in order to effectively block the metastatic cascade.\n\n\nAbbreviations\n\nCAF, carcinoma-associated fibroblasts; CITED1, Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain, 1; Cdc42, cell division control protein 42 homolog; DOCK3, dedicator of cytokinesis 3; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; FilGAP, filamin-A-associated Rho GTPase activation protein; GEF, guanine nucleotide exchange factor; GAP, GTPase activation protein; GTP, guanosine triphosphate; JAK, Janus kinase; LIF, leukemia inhibitory factor; MLC2, myosin light chain 2; MMP, matrix metallopeptidase; MRTF, myocardin-related transcription factors; PDE5A, phosphodiesterase 5A; PEDF, pigment epithelium-derived factor; Rac1, Ras-related C3 botulinum toxin substrate 1; RasGRF2, Ras protein-specific guanine nucleotide-releasing factor 2; RhoA, Ras homolog gene family member A; RhoC, Ras homolog gene family member C; ROCK, Rho-associated protein kinase; SMAD2, Sma- and Mad-related protein 2; SRF, serum response factor; STAT, signal transducer and activator of transcription; TGFβ, transforming growth factor beta.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work was supported by Cancer Research UK C33043/A12065 (Victoria Sanz-Moreno and Irene Rodriguez-Hernandez) and Royal Society RG110591 (Victoria Sanz-Moreno). Irene Rodriguez-Hernandez is supported by Fundacion Alfonso Martin Escudero, Gaia Cantelli by the Medical Research Council (C97993H), and Bruce Fanshawe by the King’s Bioscience Institute and the Guy’s and St Thomas’ Charity Prize PhD Program in Biomedical and Translational Science.\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\nValastyan S, Weinberg RA: Tumor metastasis: molecular insights and evolving paradigms. Cell. 2011; 147(2): 275–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGupta GP, Massagué J: Cancer metastasis: building a framework. Cell. 2006; 127(4): 679–95. 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PubMed Abstract | Publisher Full Text\n\nLämmermann T, Sixt M: Mechanical modes of 'amoeboid' cell migration. Curr Opin Cell Biol. 2009; 21(5): 636–44. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nGiampieri S, Manning C, Hooper S, et al.: Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive to single cell motility. Nat Cell Biol. 2009; 11(11): 1287–96. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSanz-Moreno V, Marshall CJ: The plasticity of cytoskeletal dynamics underlying neoplastic cell migration. Curr Opin Cell Biol. 2010; 22(5): 690–6. PubMed Abstract | Publisher Full Text\n\nGaggioli C, Hooper S, Hidalgo-Carcedo C, et al.: Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol. 2007; 9(12): 1392–400. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFriedl P, Wolf K, Zegers MM: Rho-directed forces in collective migration. Nat Cell Biol. 2014; 16(3): 208–10. PubMed Abstract | Publisher Full Text\n\nCharras GT, Hu CK, Coughlin M, et al.: Reassembly of contractile actin cortex in cell blebs. J Cell Biol. 2006; 175(3): 477–90. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTozluoğlu M, Tournier AL, Jenkins RP, et al.: Matrix geometry determines optimal cancer cell migration strategy and modulates response to interventions. Nat Cell Biol. 2013; 15(7): 751–62. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSahai E, Marshall CJ: Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol. 2003; 5(8): 711–9. 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PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPinner S, Sahai E: Imaging amoeboid cancer cell motility in vivo. J Microsc. 2008; 231(3): 441–5. PubMed Abstract | Publisher Full Text\n\nWyckoff JB, Pinner SE, Gschmeissner S, et al.: ROCK- and myosin-dependent matrix deformation enables protease-independent tumor-cell invasion in vivo. Curr Biol. 2006; 16(15): 1515–23. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSaito K, Ozawa Y, Hibino K, et al.: FilGAP, a Rho/Rho-associated protein kinase-regulated GTPase-activating protein for Rac, controls tumor cell migration. Mol Biol Cell. 2012; 23(24): 4739–50. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCantelli G, Orgaz JL, Rodriguez-Hernandez I, et al.: TGF-β-Induced Transcription Sustains Amoeboid Melanoma Migration and Dissemination. Curr Biol. 2015; 25(22): 2899–914. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDoyle AD, Petrie RJ, Kutys ML, et al.: Dimensions in cell migration. Curr Opin Cell Biol. 2013; 25(5): 642–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRuprecht V, Wieser S, Callan-Jones A, et al.: Cortical contractility triggers a stochastic switch to fast amoeboid cell motility. Cell. 2015; 160(4): 673–85. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nProvenzano PP, Keely PJ: Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling. J Cell Sci. 2011; 124(Pt 8): 1195–205. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPinner S, Sahai E: PDK1 regulates cancer cell motility by antagonising inhibition of ROCK1 by RhoE. Nat Cell Biol. 2008; 10(2): 127–37. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAlbrengues J, Bourget I, Pons C, et al.: LIF mediates proinvasive activation of stromal fibroblasts in cancer. Cell Rep. 2014; 7(5): 1664–78. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAlbrengues J, Bertero T, Grasset E, et al.: Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts. Nat Commun. 2015; 6: 10204. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCalvo F, Ege N, Grande-Garcia A, et al.: Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. Nat Cell Biol. 2013; 15(6): 637–46. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDel Pozo Martin Y, Park D, Ramachandran A, et al.: Mesenchymal Cancer Cell-Stroma Crosstalk Promotes Niche Activation, Epithelial Reversion, and Metastatic Colonization. Cell Rep. 2015; 13(11): 2456–69. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nReymond N, d'Água BB, Ridley AJ: Crossing the endothelial barrier during metastasis. Nat Rev Cancer. 2013; 13(12): 858–70. PubMed Abstract | Publisher Full Text\n\nHarney AS, Arwert EN, Entenberg D, et al.: Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA. Cancer Discov. 2015; 5(9): 932–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatsialou A, Bravo-Cordero JJ, Wang Y, et al.: Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors. Intravital. 2013; 2(2): e25294. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoh-Johnson M, Bravo-Cordero JJ, Patsialou A, et al.: Macrophage contact induces RhoA GTPase signaling to trigger tumor cell intravasation. Oncogene. 2014; 33(33): 4203–12. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSahai E: Illuminating the metastatic process. Nat Rev Cancer. 2007; 7(10): 737–49. PubMed Abstract | Publisher Full Text\n\nSahai E, Garcia-Medina R, Pouysségur J, et al.: Smurf1 regulates tumor cell plasticity and motility through degradation of RhoA leading to localized inhibition of contractility. J Cell Biol. 2007; 176(1): 35–42. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTremblay PL, Huot J, Auger FA: Mechanisms by which E-selectin regulates diapedesis of colon cancer cells under flow conditions. Cancer Res. 2008; 68(13): 5167–76. PubMed Abstract | Publisher Full Text\n\nEngelhardt B, Wolburg H: Mini-review: Transendothelial migration of leukocytes: through the front door or around the side of the house? Eur J Immunol. 2004; 34(11): 2955–63. PubMed Abstract | Publisher Full Text\n\nKhuon S, Liang L, Dettman RW, et al.: Myosin light chain kinase mediates transcellular intravasation of breast cancer cells through the underlying endothelial cells: a three-dimensional FRET study. J Cell Sci. 2010; 123(Pt 3): 431–40. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWorthylake RA, Lemoine S, Watson JM, et al.: RhoA is required for monocyte tail retraction during transendothelial migration. J Cell Biol. 2001; 154(1): 147–60. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHoning H, van den Berg TK, van der Pol SM, et al.: RhoA activation promotes transendothelial migration of monocytes via ROCK. J Leukoc Biol. 2004; 75(3): 523–8. PubMed Abstract | Publisher Full Text\n\nHeasman SJ, Carlin LM, Cox S, et al.: Coordinated RhoA signaling at the leading edge and uropod is required for T cell transendothelial migration. J Cell Biol. 2010; 190(4): 553–63. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nReymond N, Im JH, Garg R, et al.: RhoC and ROCKs regulate cancer cell interactions with endothelial cells. Mol Oncol. 2015; 9(6): 1043–55. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nClark EA, Golub TR, Lander ES, et al.: Genomic analysis of metastasis reveals an essential role for RhoC. Nature. 2000; 406(6795): 532–5. PubMed Abstract | Publisher Full Text\n\nReymond N, Im JH, Garg R, et al.: Cdc42 promotes transendothelial migration of cancer cells through β1 integrin. J Cell Biol. 2012; 199(4): 653–68. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nYagi H, Tan W, Dillenburg-Pilla P, et al.: A synthetic biology approach reveals a CXCR4-G13-Rho signaling axis driving transendothelial migration of metastatic breast cancer cells. Sci Signal. 2011; 4(191): ra60. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBorrull A, Ghislin S, Deshayes F, et al.: Nanog and Oct4 overexpression increases motility and transmigration of melanoma cells. J Cancer Res Clin Oncol. 2012; 138(7): 1145–54. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSabbir MG, Prieditis H, Ravinsky E, et al.: The role of Dlc1 isoform 2 in K-Ras2G12D induced thymic cancer. PLoS One. 2012; 7(7): e40302. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLi B, Zhao WD, Tan ZM, et al.: Involvement of Rho/ROCK signalling in small cell lung cancer migration through human brain microvascular endothelial cells. FEBS Lett. 2006; 580(17): 4252–60. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nGavard J, Gutkind JS: Protein kinase C-related kinase and ROCK are required for thrombin-induced endothelial cell permeability downstream from Galpha12/13 and Galpha11/q. J Biol Chem. 2008; 283(44): 29888–96. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nvan Nieuw Amerongen GP, van Delft S, Vermeer MA, et al.: Activation of RhoA by thrombin in endothelial hyperpermeability: role of Rho kinase and protein tyrosine kinases. Circ Res. 2000; 87(4): 335–40. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nZhang P, Feng S, Liu G, et al.: Mutant B-Raf(V600E) Promotes Melanoma Paracellular Transmigration by Inducing Thrombin-mediated Endothelial Junction Breakdown. J Biol Chem. 2016; 291(5): 2087–106. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nNierodzik ML, Karpatkin S: Thrombin induces tumor growth, metastasis, and angiogenesis: Evidence for a thrombin-regulated dormant tumor phenotype. Cancer Cell. 2006; 10(5): 355–62. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRiento K, Ridley AJ: Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol. 2003; 4(6): 446–56. PubMed Abstract | Publisher Full Text\n\nWysolmerski RB, Lagunoff D: Involvement of myosin light-chain kinase in endothelial cell retraction. Proc Natl Acad Sci U S A. 1990; 87(1): 16–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGroeger G, Nobes CD: Co-operative Cdc42 and Rho signalling mediates ephrinB-triggered endothelial cell retraction. Biochem J. 2007; 404(1): 23–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHuang B, Krafft PR, Ma Q, et al.: Fibroblast growth factors preserve blood-brain barrier integrity through RhoA inhibition after intracerebral hemorrhage in mice. Neurobiol Dis. 2012; 46(1): 204–14. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nFujii M, Duris K, Altay O, et al.: Inhibition of Rho kinase by hydroxyfasudil attenuates brain edema after subarachnoid hemorrhage in rats. Neurochem Int. 2012; 60(3): 327–33. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGajewski TF, Schreiber H, Fu YX: Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol. 2013; 14(10): 1014–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMedjkane S, Perez-Sanchez C, Gaggioli C, et al.: Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis. Nat Cell Biol. 2009; 11(3): 257–68. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nOrgaz JL, Ladhani O, Hoek KS, et al.: 'Loss of pigment epithelium-derived factor enables migration, invasion and metastatic spread of human melanoma'. Oncogene. 2009; 28(47): 4147–61. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGarcia M, Fernandez-Garcia NI, Rivas V, et al.: Inhibition of xenografted human melanoma growth and prevention of metastasis development by dual antiangiogenic/antitumor activities of pigment epithelium-derived factor. Cancer Res. 2004; 64(16): 5632–42. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLadhani O, Sánchez-Martinez C, Orgaz JL, et al.: Pigment epithelium-derived factor blocks tumor extravasation by suppressing amoeboid morphology and mesenchymal proteolysis. Neoplasia. 2011; 13(7): 633–42. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nArozarena I, Sanchez-Laorden B, Packer L, et al.: Oncogenic BRAF induces melanoma cell invasion by downregulating the cGMP-specific phosphodiesterase PDE5A. Cancer Cell. 2011; 19(1): 45–57. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nCalvo F, Sanz-Moreno V, Agudo-Ibáñez L, et al.: RasGRF suppresses Cdc42-mediated tumour cell movement, cytoskeletal dynamics and transformation. Nat Cell Biol. 2011; 13(7): 819–26. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPadua D, Zhang XH, Wang Q, et al.: TGFbeta primes breast tumors for lung metastasis seeding through angiopoietin-like 4. Cell. 2008; 133(1): 66–77. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nThiery JP: Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer. 2002; 2(6): 442–54. PubMed Abstract | Publisher Full Text\n\nRedondo-Muñoz J, Ugarte-Berzal E, Terol MJ, et al.: Matrix metalloproteinase-9 promotes chronic lymphocytic leukemia b cell survival through its hemopexin domain. Cancer Cell. 2010; 17(2): 160–72. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nGould SE, Junttila MR, de Sauvage FJ: Translational value of mouse models in oncology drug development. Nat Med. 2015; 21(5): 431–9. PubMed Abstract | Publisher Full Text\n\nKircher MF, Gambhir SS, Grimm J: Noninvasive cell-tracking methods. Nat Rev Clin Oncol. 2011; 8(11): 677–88. PubMed Abstract | Publisher Full Text\n\nBrader P, Serganova I, Blasberg RG: Noninvasive molecular imaging using reporter genes. J Nucl Med. 2013; 54(2): 167–72. PubMed Abstract | Publisher Full Text\n\nLawler K, Foran E, O'Sullivan G, et al.: Mobility and invasiveness of metastatic esophageal cancer are potentiated by shear stress in a ROCK- and Ras-dependent manner. Am J Physiol Cell Physiol. 2006; 291(4): C668–77. PubMed Abstract | Publisher Full Text\n\nReymond N, Riou P, Ridley AJ: Rho GTPases and cancer cell transendothelial migration. Methods Mol Biol. 2012; 827: 123–42. PubMed Abstract | Publisher Full Text\n\nParri M, Taddei ML, Bianchini F, et al.: EphA2 reexpression prompts invasion of melanoma cells shifting from mesenchymal to amoeboid-like motility style. Cancer Res. 2009; 69(5): 2072–81. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13658",
"date": "29 Apr 2016",
"name": "Martin A. Schwartz",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13659",
"date": "29 Apr 2016",
"name": "Michael Olson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-783
|
https://f1000research.com/articles/5-782/v1
|
29 Apr 16
|
{
"type": "Review",
"title": "Recent advances in large-scale protein interactome mapping",
"authors": [
"Virja Mehta",
"Laura Trinkle-Mulcahy",
"Virja Mehta"
],
"abstract": "Protein-protein interactions (PPIs) underlie most, if not all, cellular functions. The comprehensive mapping of these complex networks of stable and transient associations thus remains a key goal, both for systems biology-based initiatives (where it can be combined with other ‘omics’ data to gain a better understanding of functional pathways and networks) and for focused biological studies. Despite the significant challenges of such an undertaking, major strides have been made over the past few years. They include improvements in the computation prediction of PPIs and the literature curation of low-throughput studies of specific protein complexes, but also an increase in the deposition of high-quality data from non-biased high-throughput experimental PPI mapping strategies into publicly available databases.",
"keywords": [
"Interactome",
"Proteomics",
"AP-MS",
"affinity purification-mass spectrometry",
"XL-MS",
"cross-linking MS analysis",
"PCP",
"protein correlation profiling",
"BioID",
"APEX"
],
"content": "\n\nA range of complementary approaches are currently being used to identify protein-protein interactions (PPIs) in a large-scale, high-throughput manner (Figure 1). These include affinity purification-mass spectrometry (AP-MS), cross-linking MS (XL-MS) analysis, MS-based protein correlation profiling (PCP), and yeast two-hybrid (Y2H) screens. Proximity labeling techniques, based on the identification (by AP-MS) of near neighbor proteins by spatially restricted enzymes, can also be used to map protein networks and probe complex structures, although they have yet to be applied at a whole proteome level. In this review, we discuss recent applications of these diverse methods to large-scale protein interactome mapping and the public availability of the resulting datasets for both high-throughput bioinformatic analysis of protein interaction networks and single-protein information for more focused studies.\n\na. Affinity purification-mass spectrometry approach for identifying proteins that associate with a particular bait protein. b. Two spatially-restricted “near neighbor labeling” approaches that utilize enzymatic reactions to tag proteins (for capture and identification) that associate with a bait protein. c. Strategy behind cross-linking mass spectrometry analysis of multiprotein complexes. d. Protein correlation profiling approach for identifying multiprotein complex members that co-elute following various separation techniques. e. Strategy behind the classic yeast two-hybrid method used to screen for direct protein-protein interactions.\n\n\nAffinity purification-mass spectrometry-based large-scale protein-protein interaction mapping initiatives\n\nCurrently, the most popular strategy for both high- and low-throughput interactome mapping is AP-MS, in which an endogenous or tagged bait protein is depleted from cell lysates by using an affinity resin and associated proteins identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Figure 1a). Two recent large-scale studies of human PPIs used AP-MS approaches to identify more than 20,000 interactions, respectively (Table 1). To assemble what they call the BioPlex (biophysical interactions of ORFeome-derived complexes), Huttlin and colleagues C-terminally FLAG-HA tagged about 600 human open reading frames (ORFs) and transiently overexpressed them in HEK293T cells, identifying co-precipitating proteins by AP-MS1. Clone validation, quality control, inclusion of positive and negative controls, and development of a quantitation algorithm (CompPASS-Plus) based on abundance, detection frequency, and reproducibility were employed to increase confidence in the resulting dataset, which was deposited into the BioGRID PPI database last year. The authors consider this to be phase 1 of their long-term effort to map interactomes for the entire human ORFeome collection and are continuing to post updates that can be freely browsed or downloaded from their website.\n\nAP-MS, affinity purification-mass spectrometry; BAC, bacterial artificial chromosome; CORUM, Comprehensive Resource of Mammalian protein complexes; GFP, green fluorescent protein; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MS, mass spectrometry; ORF, open reading frame; XL-MS; cross-linking mass spectrometry; Y2H, yeast two-hybrid.\n\nThe approach used by Hein and colleagues2 involved screening a library of 1,125 HeLa cell lines with stably incorporated N- and C-terminally tagged mouse and human bacterial artificial chromosome (BAC) transgenes under near endogenous control3 by AP-MS, as demonstrated previously in focused studies analyzing chromosome segregation4 and the function of motor proteins5. In addition to identifying more than 28,000 interactions in their large-scale screen, the authors estimated interaction stoichiometries (based on absolute quantitation of protein abundances in complexes and compared for both N- and C-terminally tagged and mouse and human bait proteins) and measured the relative cellular abundances of interaction partners. An interesting finding was the predominance of weak (i.e., sub-stoichiometric) interactions in the global interactome, which may suggest that stable complexes rely on weak links to connect to each other and to transient/dynamic regulators. The interaction datasets were submitted to both the IntAct database and the IMEx consortium.\n\nImportantly, both studies demonstrated significant overlap with the CORUM (Comprehensive Resource of Mammalian protein complexes) database, a manually curated repository of more than 2,800 mammalian protein complexes6. CORUM is currently considered the “gold standard” PPI database because it is based solely on high-confidence, experimentally verified interactions and does not accept deposition of large-scale datasets (Table 2). Proteome coverage was also high for both studies, as assessed by comparison with datasets generated and shared in recent large-scale whole proteome mapping initiatives (Table 3) such as the MaxQuant Database7–9 (MaxQB), the Human Proteome Map10, and ProteomicsDB11.\n\nAlthough the standard caveats of AP-MS strategies still apply, namely the potential for overexpression or tag-induced artefacts and the predominance of false positives such as non-specific background proteins12–14 and the recently described cryptic protein binding to cloning regions or “scars” where affinity tags are linked to the gene of interest15, these large-scale studies benefit tremendously from the comparison of multiple experiments. Negative controls are largely bait-independent, and thus common contaminants are highlighted by their appearance in numerous unrelated datasets. Moving forward, the limitations of AP-MS can be further minimized by a variety of strategies, including direct affinity tagging of endogenous proteins using the powerful CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) gene editing tool16,17, more rigorous assessment of the quality and specificity of antibodies used to capture endogenous proteins for AP-MS18, and improvements in significance analysis software19,20.\n\n\nProximity-based labeling strategies\n\nAlthough AP-MS remains the most commonly used technique for mapping PPIs, its Achilles heel has always been the necessity to break cells open to extract complexes for analysis, which can be disruptive to the underlying PPIs and hinder identification of weak or transient associations or both. The development of complementary proximity labeling approaches that use spatially restricted enzymes to biotinylate neighboring proteins has helped to address this key issue. Complex members are labeled covalently in vivo, thus eliminating the need for low-stringency purification strategies to preserve their integrity. Furthermore, the high affinity of streptavidin for biotin facilitates efficient recovery of biotinylated proteins from lysates for MS analysis.\n\nTwo particular proximity labeling techniques, BioID and APEX, have been employed recently for the analysis of multiprotein complexes and for identification of the protein components of specific cellular compartments (Figure 1b). BioID involves expression of a protein of interest fused to a prokaryotic biotin ligase and the subsequent biotinylation of amine groups on neighboring proteins when excess biotin is added to the cells. Whereas the wild-type BirA biotin ligase from Escherichia coli is capable of transferring biotin only to a substrate bearing a specific recognition sequence, the generation of a promiscuous BirA (Arg118Gly mutant) permits the biotinylation of any protein found within a 10-nm labeling radius21,22. As with AP-MS, identification of a protein-protein association using BioID does not imply a direct physical interaction.\n\nBioID has enabled the identification of proteins involved in important functional complexes that were previously difficult to characterize because of the limitations of AP-MS. For example, the identification of ubiquitin ligase substrates by AP-MS is challenging and this is due in part to the weak and transient interactions observed between the ligase and its substrates. A BioID approach, however, facilitated identification of novel substrates23. This type of approach has also been used to identify novel c-MYC24 and HIV-1 Gag25 interacting partners, highlight force-dependent molecular interactions at cell-cell adhesions26, identify proteins localized to cell junction complexes27,28 and the centrosome-cilium interface29, and probe the structure of the centrosome30,31 and the nuclear pore complex22.\n\nAPEX is a monomeric peroxidase reporter derived from pea32 or soybean33 ascorbate peroxidase that catalyzes the oxidation of biotin-phenol to biotin-phenoxyl in the presence of H2O2, resulting in the biotinylation of proteins in the neighboring region. Whereas BirA-catalyzed biotinylation is limited to Lys residues, biotin-phenoxyl radicals can covalently react with electron-rich amino acids such as Tyr, Trp, His, and Cys. They are also short-lived (<5 ms) and membrane-impermeable and have a small labeling radius (<20 nm). APEX can also catalyze diaminobenzidine precipitation to generate contrast after OsO4 fixation, which allows confirmation of localization at nanometer resolution by electron microscopy32. A second-generation APEX2 (Ala134Pro mutant) with improved efficiency was shown to function even better as both a promiscuous labeling enzyme and an EM tag34. Similar to BioID, once proximity labeling has been achieved, biotinylated proteins can be identified via stringent streptavidin purification and MS analysis. An advantage of APEX over BioID is higher temporal resolution, as labeling is achieved on a minute rather than an hour scale.\n\nThe APEX reporter has been used to map the proteome of the human mitochondrial intermembrane space and membrane-enclosed mitochondrial matrix33,35, the Drosophila muscle mitochondrial matrix proteome36, and the proteome of the cilium37. Although the applicability of APEX to interactome mapping out with membrane-bound organelles has not yet been demonstrated, further optimization of the enzyme and substrate could extend its utility.\n\n\nLarge-scale protein-protein interaction mapping initiatives based on alternative approaches\n\nHigh-quality large-scale interactome datasets have also been assembled using strategies such as XL-MS, which provides additional information about the topographical structure of protein complexes (Figure 1c and Table 1). In the case of XL-MS, progress was initially slowed by the complexity of data acquisition and analysis, in particular the two overlapping series of fragment ions from each peptide that appear in the MS/MS spectrum. Although major advances have been made38,39, including the development of MS-cleavable cross-linkers that fragment efficiently in the MS/MS mode to yield two major fragment ions corresponding to the component peptides (which can be subsequently identified by MS3), sensitivity can be further improved in the future by the addition of pre-fractionation steps, the use of affinity-tagged cross-linking agents or complementary chemistry (i.e., agents that cross-link amino acids other than lysine40), digestion with complementary proteases, and the development of dedicated software for the analysis of complex XL-MS datasets.\n\nSimilarly, PCP-MS studies (Figure 1d) also continue to increase in coverage and specificity, comparing favorably to reference interactome datasets41–43. This approach avoids affinity purification steps and instead separates and maps protein complexes using a variety of approaches that include density gradients and size-exclusion, ion-exclusion, and hydrophobicity interaction chromatography. Given the range of separation options available, PCP-MS also offers significant scope for advancement in the future.\n\n\nLarge-scale binary protein-protein interaction mapping\n\nAlthough XL-MS does identify direct protein interactions, the other approaches discussed above (AP-MS, proximity labeling, and PCP-MS) can confirm only that proteins exist in the same multiprotein complex. A complementary technique that has been used for more than 20 years to detect direct PPI is the Y2H assay. In this approach, the bait and prey proteins are tagged to the DNA binding and transcriptional activation domains of a split transcription factor, and direct binding drives its reconstitution and subsequent activation of a reporter gene (Figure 1e). Although limited by technical and biological challenges that include the need to construct large libraries and the high false-negative and -positive rates that arise from the absence of certain post-translational modifications in yeast that govern protein-protein associations in mammalian cells and forced interactions that do not occur in mammalian cells under physiological conditions, the Y2H screen remains a powerful approach for detecting or confirming (or both) binary interactions.\n\nUsing the extensive human ORF collection as bait/prey in an ongoing series of large-scale Y2H screens, researchers at the Dana-Farber Cancer Institute in Boston are addressing the question of which PPIs in the human interactome are direct44,45. With the long-term goal of mapping the full range of human binary PPIs, their most recent update added about 14,000 new binary interactions, bringing the current total to about 17,000. The full dataset, and future updates, can be browsed using their dedicated web interface (Table 1).\n\n\nConclusions\n\nWith a daunting grand plan for these PPI network maps to comprehensively characterize individual protein functions and global proteome organization, it is not surprising that significant challenges remain. As noted above, the stringency and efficiency of protein extraction and depletion remain an issue with AP-MS studies, and traditional mapping strategies still favor the most abundant/robust interactors. It is hoped that, as complementary approaches such as proximity labeling, XL-MS, and PCP-MS increase in sensitivity and specificity, they will provide extended coverage of the interactome. Importantly, as more high-quality large-scale datasets are collected and shared via online interaction databases like MIntAct46 and BioGRID47 (Table 2), consistencies and patterns will emerge.\n\nAdditional technical challenges, posed by their hydrophobic nature, have particularly hampered the identification of PPIs among membrane proteins (and between membrane proteins and soluble proteins such as cytosolic signaling factors). However, the success of recent large-scale initiatives such as the mapping of more than 12,000 binary interactions between Arabidopsis membrane/signaling proteins using the mating-based split ubiquitin system (mbSUS) in yeast48 and the TAP (tandem affinity purification)-MS based screening of 1,590 putative budding yeast membrane proteins using three different mild, non-denaturing detergent purification strategies in parallel49 (1,726 PPIs and 501 putative heteromeric complexes identified) demonstrates that these challenges are also surmountable.\n\nOther challenges include the necessity to define PPIs over a wider range of biological contexts, given that some are cell cycle- or developmental stage-specific, for example, or occur only under particular physiological conditions or in response to specific post-translational modifications. An ambitious future goal is a comprehensive and quantitative high-throughput approach that combines gene-editing with live super-resolution imaging and interactome mapping to define the dynamic localization, composition, and topography of functional multiprotein complexes.\n\n\nAbbreviations\n\nAP-MS, affinity purification-mass spectrometry; CORUM, Comprehensive Resource of Mammalian protein complexes; ORF, open reading frame; PCP, protein correlation profiling; PPI, protein-protein interaction; XL-MS; cross-linking mass spectrometry; Y2H, yeast two-hybrid.",
"appendix": "Author contributions\n\n\n\nVirja Mehta and Laura Trinkle-Mulcahy wrote the manuscript. Both authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHuttlin EL, Ting L, Bruckner RJ, et al.: The BioPlex Network: A Systematic Exploration of the Human Interactome. Cell. 2015; 162(2): 425–40. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHein MY, Hubner NC, Poser I, et al.: A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell. 2015; 163(3): 712–23. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPoser I, Sarov M, Hutchins JR, et al.: BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat Methods. 2008; 5(5): 409–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHutchins JR, Toyoda Y, Hegemann B, et al.: Systematic analysis of human protein complexes identifies chromosome segregation proteins. Science. 2010; 328(5978): 593–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMaliga Z, Junqueira M, Toyoda Y, et al.: A genomic toolkit to investigate kinesin and myosin motor function in cells. Nat Cell Biol. 2013; 15(3): 325–34. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRuepp A, Waegele B, Lechner M, et al.: CORUM: the comprehensive resource of mammalian protein complexes--2009. Nucleic Acids Res. 2010; 38(Database issue): D497–501. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKulak NA, Pichler G, Paron I, et al.: Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods. 2014; 11(3): 319–24. PubMed Abstract | Publisher Full Text\n\nGeiger T, Wehner A, Schaab C, et al.: Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins. Mol Cell Proteomics. 2012; 11(3): M111.014050. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchaab C, Geiger T, Stoehr G, et al.: Analysis of high accuracy, quantitative proteomics data in the MaxQB database. Mol Cell Proteomics. 2012; 11(3): M111.014068. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim MS, Pinto SM, Getnet D, et al.: A draft map of the human proteome. Nature. 2014; 509(7502): 575–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilhelm M, Schlegl J, Hahne H, et al.: Mass-spectrometry-based draft of the human proteome. Nature. 2014; 509(7502): 582–7. PubMed Abstract | Publisher Full Text\n\nMellacheruvu D, Wright Z, Couzens AL, et al.: The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods. 2013; 10(8): 730–6. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBoulon S, Ahmad Y, Trinkle-Mulcahy L, et al.: Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol Cell Proteomics. 2010; 9(5): 861–79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTrinkle-Mulcahy L, Boulon S, Lam YW, et al.: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol. 2008; 183(2): 223–39. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBanks CA, Boanca G, Lee ZT, et al.: Proteins interacting with cloning scars: a source of false positive protein-protein interactions. Sci Rep. 2015; 5: 8530. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCong L, Ran FA, Cox D, et al.: Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339(6121): 819–23. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRatz M, Testa I, Hell SW, et al.: CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells. Sci Rep. 2015; 5: 9592. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMarcon E, Jain H, Bhattacharya A, et al.: Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation. Nat Methods. 2015; 12(8): 725–31. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nTeo G, Liu G, Zhang J, et al.: SAINTexpress: improvements and additional features in Significance Analysis of INTeractome software. J Proteomics. 2014; 100: 37–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorris JH, Knudsen GM, Verschueren E, et al.: Affinity purification-mass spectrometry and network analysis to understand protein-protein interactions. Nat Protoc. 2014; 9(11): 2539–54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoux KJ, Kim DI, Raida M, et al.: A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol. 2012; 196(6): 801–10. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKim DI, Birendra KC, Zhu W, et al.: Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proc Natl Acad Sci U S A. 2014; 111(24): E2453–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCoyaud E, Mis M, Laurent EM, et al.: BioID-based Identification of Skp Cullin F-box (SCF)β-TrCP1/2 E3 Ligase Substrates. Mol Cell Proteomics. 2015; 14(7): 1781–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDingar D, Kalkat M, Chan PK, et al.: BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors. J Proteomics. 2015; 118: 95–111. PubMed Abstract | Publisher Full Text\n\nLe Sage V, Cinti A, Valiente-Echeverría F, et al.: Proteomic analysis of HIV-1 Gag interacting partners using proximity-dependent biotinylation. Virol J. 2015; 12: 138. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUeda S, Blee AM, Macway KG, et al.: Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase. PLoS One. 2015; 10(3): e0122886. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFredriksson K, Van Itallie CM, Aponte A, et al.: Proteomic analysis of proteins surrounding occludin and claudin-4 reveals their proximity to signaling and trafficking networks. PLoS One. 2015; 10(3): e0117074. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVan Itallie CM, Aponte A, Tietgens AJ, et al.: The N and C termini of ZO-1 are surrounded by distinct proteins and functional protein networks. J Biol Chem. 2013; 288(19): 13775–88. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGupta GD, Coyaud É, Gonçalves J, et al.: A Dynamic Protein Interaction Landscape of the Human Centrosome-Cilium Interface. Cell. 2015; 163(6): 1484–99. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFirat-Karalar EN, Rauniyar N, Yates JR 3rd, et al.: Proximity interactions among centrosome components identify regulators of centriole duplication. Curr Biol. 2014; 24(6): 664–70. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nFirat-Karalar EN, Stearns T: Probing mammalian centrosome structure using BioID proximity-dependent biotinylation. Methods Cell Biol. 2015; 129: 153–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartell JD, Deerinck TJ, Sancak Y, et al.: Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol. 2012; 30(11): 1143–8. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRhee HW, Zou P, Udeshi ND, et al.: Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science. 2013; 339(6125): 1328–31. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLam SS, Martell JD, Kamer KJ, et al.: Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods. 2015; 12(1): 51–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHung V, Zou P, Rhee HW, et al.: Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging. Mol Cell. 2014; 55(2): 332–41. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nChen CL, Hu Y, Udeshi ND, et al.: Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase. Proc Natl Acad Sci U S A. 2015; 112(39): 12093–8. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMick DU, Rodrigues RB, Leib RD, et al.: Proteomics of Primary Cilia by Proximity Labeling. Dev Cell. 2015; 35(4): 497–512. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLiu F, Rijkers DT, Post H, et al.: Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry. Nat Methods. 2015; 12(12): 1179–84. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWalzthoeni T, Joachimiak LA, Rosenberger G, et al.: xTract: software for characterizing conformational changes of protein complexes by quantitative cross-linking mass spectrometry. Nat Methods. 2015; 12(12): 1185–90. PubMed Abstract | Publisher Full Text\n\nLeitner A, Joachimiak LA, Unverdorben P, et al.: Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes. Proc Natl Acad Sci U S A. 2014; 111(26): 9455–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKirkwood KJ, Ahmad Y, Larance M, et al.: Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics. Mol Cell Proteomics. 2013; 12(12): 3851–73. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHavugimana PC, Hart GT, Nepusz T, et al.: A census of human soluble protein complexes. Cell. 2012; 150(5): 1068–81. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKristensen AR, Gsponer J, Foster LJ: A high-throughput approach for measuring temporal changes in the interactome. Nat Methods. 2012; 9(9): 907–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRual JF, Venkatesan K, Hao T, et al.: Towards a proteome-scale map of the human protein-protein interaction network. Nature. 2005; 437(7062): 1173–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRolland T, Taşan M, Charloteaux B, et al.: A proteome-scale map of the human interactome network. Cell. 2014; 159(5): 1212–26. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nOrchard S, Ammari M, Aranda B, et al.: The MIntAct project--IntAct as a common curation platform for 11 molecular interaction databases. Nucleic Acids Res. 2014; 42(Database issue): D358–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChatr-Aryamontri A, Breitkreutz BJ, Oughtred R, et al.: The BioGRID interaction database: 2015 update. Nucleic Acids Res. 2015; 43(Database issue): D470–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones AM, Xuan Y, Xu M, et al.: Border control--a membrane-linked interactome of Arabidopsis. Science. 2014; 344(6185): 711–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBabu M, Vlasblom J, Pu S, et al.: Interaction landscape of membrane-protein complexes in Saccharomyces cerevisiae. Nature. 2012; 489(7417): 585–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "13655",
"date": "29 Apr 2016",
"name": "Francois-Michel Boisvert",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13656",
"date": "29 Apr 2016",
"name": "Arnaud Poterszman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13657",
"date": "29 Apr 2016",
"name": "Christiane Schaffitzel",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-782
|
https://f1000research.com/articles/5-781/v1
|
29 Apr 16
|
{
"type": "Opinion Article",
"title": "Time for sharing data to become routine: the seven excuses for not doing so are all invalid",
"authors": [
"Richard Smith",
"Ian Roberts",
"Ian Roberts"
],
"abstract": "Data are more valuable than scientific papers but researchers are incentivised to publish papers not share data. Patients are the main beneficiaries of data sharing but researchers have several incentives not to share: others might use their data to get ahead in the academic rat race; they might be scooped; their results might not be replicable; competitors may reach different conclusions; their data management might be exposed as poor; patient confidentiality might be breached; and technical difficulties make sharing impossible. All of these barriers can be overcome and researchers should be rewarded for sharing data. Data sharing must become routine.",
"keywords": [
"Data sharing",
"data analysis",
"data management",
"publishing"
],
"content": "\n\nGood, well curated data are more valuable than the words authors write about them, but until now the main currency of science has been publications. With the World Wide Web sharing and publishing data is now possible, and researchers should be rewarded for doing so. Authors unfortunately have incentives not to share data and continue to find excuses for not doing so – but the excuses are poor. It’s time for data sharing to become routine.\n\n\nThe value of data\n\nDatasets are more valuable than papers because: they allow analyses to be replicated helping to avoid error, selective reporting and fraud; they can be used to answer other research questions; and they facilitate methodological research and the teaching and training of researchers. Papers, in contrast, rarely report the full data and are often “spun” to present results that flatter authors and please editors.\n\n\nPatients are the main beneficiaries of data sharing\n\nThe main beneficiaries of sharing data are patients, the people who as taxpayers fund most research. They clearly have an interest in both the right conclusion being reached and in maximum value being squeezed from every dataset. Unfortunately many others in the research system do not have the same interest in the “truth.”\n\nIf we consider a clinical trial or indeed any study with clinical implications then the prime interest of the patients is that the results are “true” and that clinicians use them to improve their well-being. This means that the analyses should be accurate and replicable. Sadly the producers of research have interests apart from truth: researchers want high impact papers; universities want the same and lots of publicity too; editors and publishers want “good” publications that increase their impact factor; and funders want to show “value for money,” which may means lots of publications regardless of their truth. Nobody is incentivised to share data, replicate results, and perhaps show the weak underbelly of science, which is why the scientific community has responded so poorly to allegations of misconduct1.\n\nBy participating in clinical research patients make a gift to others, rather as those who give blood do. They and their gift, their data, should be treated with reverence. Their gift is not for individual researchers to use to advance their careers but for the wider scientific community and other patients. Their gift must be shared.\n\n\nThe seven incentives not to share\n\nBecause they are measured primarily by how much and where they publish, researchers are strongly incentivised to publish, preferably in high impact journals. There are not the same incentives to share data. Indeed, there are seven incentives (or excuses) not to share.\n\nFirstly, data are the base for research articles, and one anxiety for researchers is that others will use their data to produce publications without having to go to the trouble of gathering them. They will be disadvantaged in the academic rat race, although if everybody shared data they could benefit from using data from others.\n\nSecondly, other researchers might scoop them, perhaps even prevent them from achieving publication in a high impact journal. Funders who require data sharing have responded to the anxiety of being scooped by allowing researchers to delay sharing their data. A better response would be to move away from “outsourcing” the judgement of the performance of researchers to publishers and for employers and funders to recognise that judging researchers is core business that should not be outsourced to the arbitrary and corrupted publishing process.\n\nA third reason for not sharing data is a fear held by researchers that their conclusions will not be replicable. This is an ignoble reason because replicability is central to science. Some scientists may fear replication because they repeat experiments day after day and publish them only when they become “right.” This is unscientific and can lead to serious defects in the scientific evidence base.\n\nOne of us (IR) has made data from two large clinical trials available in the hope that somebody will replicate the analysis and confirm (or fail to confirm) the results (https://ctu-app.lshtm.ac.uk/freebird/)2,3. Although the data have been used to answer many different questions, there has been no replication of the original trial results, probably because there is no incentive to do so - there ought to be. It surely makes economic sense for the millions spent on the trial to be backed up by the few thousands that would be needed to encourage replication. We hope that somebody will take up the challenge.\n\nA fourth reason researchers may want to keep their data to themselves is to avoid their critics analysing the data and coming up with different or contrary results. Statisticians say that “if you torture the data they will confess,” but refusing to release data hands a victory to critics who will inevitably say “the researchers obviously have something to hide, they can’t support their conclusions.” Uncomfortable as it may be, it’s a better and more scientific strategy to enter “the market of ideas” and expect to show the correctness of your analysis and conclusions.\n\nThere is a legitimate worry about releasing data when researchers fear they may be sued. The problem here is that a battle in court is not a battle of evidence and data but a battle of showmen with a highly uncertain outcome. This is not a worry with most datasets, and perhaps when it is the data can be released in exchange for a legally binding commitment not to sue.\n\nThe authors of a major trial that showed the ineffectiveness of hydroxyethyl starch solutions for fluid resuscitation have declined to share their data4,5. They say that there have been “repeated efforts to discredit” by critics who want “to protect their commercial interests.” The authors have declined even to allow a reanalysis by a third party. This cannot be in the interest of patients, who clearly want to know whether the treatment is ineffective or not, but the authors may have a legitimate worry about legal action.\n\nThe fifth and perhaps worst reason for not releasing data is that data management is often poor and sharing the data may expose horrible weaknesses, flaws, and inconsistencies in the data. Sadly this may be the commonest but least declared reason for not sharing data. That some universities dedicate more resources to media relations than research governance is disturbing but not surprising. Making a big splash in the news can bolster grant income and student recruitment even when the informational content of the research is doubtful.\n\nA sixth excuse for not sharing data that is available to those who do research with patients is patient confidentiality. One case of private information of a patient being exposed could, some researchers argue, bring data sharing to a halt. It is a “never event” that must be avoided even if huge benefits are foregone by not sharing data. Patient confidentiality must be guarded, and most of the time it’s easy to do so by anonymising data and removing data on, for example, place and time. It’s true that small risks remain because of rare conditions and events and because of “jigsawing” (combining datasets to break confidentiality), but these small risks can be explained to patients, who will almost always consent to their data being made available in anonymous form. With datasets that are already collected patients might be asked to give retrospective consent.\n\nPatient confidentiality is the reason that authors of a controversial trial on treatment of chronic fatigue syndrome give for not sharing their data, but inevitably they look as if they are hiding something6,7.\n\nThe final and probably weakest excuse researchers give for not sharing data is “technical reasons.” But this is a lame excuse—other areas of science—for example, physics, astronomy, and engineering—have shared datasets far larger and more complex than those produced in biomedical research. There are no insurmountable technical reasons to sharing and publishing data.\n\n\nReward authors for sharing data\n\nResearchers should be rewarded not for publications but for producing large amounts of high quality data. Papers are a poor measure of the quantity or quality of research data. In terms of papers, a trial with 100 patients is the same as one with 10 000 patients, even though the informational content of the latter is 100 times the former. And despite the reverence for peer review, data quality is remarkably hard to judge from publications.\n\nFunders of research and employers of researchers need to change the incentives for researchers to encourage data sharing, but researchers must also recognise the weakness of their excuses and contribute to the big advance in science that can come from sharing and publishing data.",
"appendix": "Author contributions\n\n\n\nBoth authors contributed to the paper and have read and approved the final version.\n\n\nCompeting interests\n\n\n\nRS is a paid consultant to F1000Research, which requires submission of full data with research articles. IR works at LSHTM which received NIHR funds to set up a data sharing website (https://ctu-app.lshtm.ac.uk/freebird/).\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSmith R: Statutory regulation needed to expose and stop medical fraud. BMJ. 2016; 352: i293. PubMed Abstract | Publisher Full Text\n\nThe CRASH-2 trial collaborators, Shakur H, Roberts I, et al.: Effects of tranexamic acid on death, vascular occlusive events, and blood transfusion in trauma patients with significant haemorrhage (CRASH-2): a randomised, placebo-controlled trial. Lancet. 2010; 376(9734): 23–32. PubMed Abstract | Publisher Full Text\n\nEdwards P, Arango M, Balica L, et al.: Final results of MRC CRASH, a randomised placebo-controlled trial of intravenous corticosteroid in adults with head injury-outcomes at 6 months. Lancet. 2005; 365(9475): 1957–9. PubMed Abstract | Publisher Full Text\n\nDoshi P: Data too important to share: do those who control the data control the message? BMJ. 2016; 352: i1027. PubMed Abstract | Publisher Full Text\n\nMyburgh JA, Finfer S, Bellomo R, et al.: Hydroxyethyl starch or saline for fluid resuscitation in intensive care. N Engl J Med. 2012; 367(20): 1901–11. PubMed Abstract | Publisher Full Text\n\nSmith R: QMUL and King’s college should release data from the PACE trial. Reference Source\n\nWhite PD, Goldsmith KA, Johnson AL, et al.: Comparison of adaptive pacing therapy, cognitive behaviour therapy, graded exercise therapy, and specialist medical care for chronic fatigue syndrome (PACE): a randomised trial. Lancet. 2011; 377(9768): 823–36. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "13665",
"date": "05 May 2016",
"name": "Tom Walley",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nData sharing has been an expectation and indeed a contractual obligation for all research funded by NIHR, the research arm of the NHS, for many years. This has meant that bona fides researchers can request access to study data for defined proposes and with a suitable protocol, which should not be unreasonably withheld, e.g. for purposes of IPD meta-analysis. This is not open but controlled access to the data. The arbiter of what is reasonable access to the data falls to the researcher in the first instance, then to his/her host institute, but ultimately to the funder who held the contract. The recent consultation from the ICMJE (http://www.nejm.org/doi/full/10.1056/NEJMe1515172 will probably translate into a requirement that data sets be made available in a more transparent way, usually by host institutions, in some form of as yet undefined registry.Why not open access? Smith and Roberts consider some of these issues:Ownership of the data: this (and responsibility for curation and archiving) rests with the institute but subject to the terms of the contract. Inevitably however, a researcher will feel a degree of proprietary protectiveness towards data sets. Most of us are not as altruistic in this regard as Smith and Roberts would like. Given the incentives that exist in academia, some respect for the intellectual property that the researcher has created is inevitable, and usually an agreement to access the data either in collaboration or with due acknowledgement is an acceptable outcome for all.Risks of confidentiality: many studies are not of the 20000 patients size that Roberts has made available: smaller studies, with geographically defined recruitment may mean that the patient is potentially identifiable, especially if complex sets of data – often collected in smaller studies but less likely in larger - can also be accessed. Regrettably, there are people who seem to thrive on breaking open data like this: I think that patient confidentiality requires us to ensure that the data remains anonymous, best achieved by limited rather than open access.Poor data handling: making data available to others is not without substantial cost, at a time when most researchers are planning to move on to another study: e.g. labelling the files from complex data sets in clear manner understandable to those who have not lived and breathed it for several years. Hence collaborative access is an easier and less expensive solution, where possible. Archiving the data also poses problems – who will take responsibility for converting data from old systems or software.NIHR have established a contractual obligation, but like most other funders, has not yet provided the level of funding to make this possible (except on one occasion to Roberts), nor a vehicle similar to the GSK-led clinicalstudydatarequest.com to facilitate this.None of this is to argue against the principles that Smith and Roberts put forward, but only to point out that achieving their worthy aims will not be easy or as quick as it might seem. NIHR like other funders continue to work to support this aim. As part of this, the NIHR journals library is also considering what constitutes publication: perhaps a somewhat selective journal article, a detailed monograph as has been our practice (www.journalslibrary.nihr.ac.uk) or in the future, such a document with access to the data. These questions will not be quickly solved, and need much more debate to which this article by Smith and Roberts is a valuable contribution",
"responses": [
{
"c_id": "1972",
"date": "11 May 2016",
"name": "Richard Smith",
"role": "Author Response F1000Research Advisory Board Member",
"response": "I’m grateful to Tom for giving a rapid and useful on our paper. No doubt he is right that it will take a longer time than we would like for data sharing to become routine.Incentives are fundamental. At the moment incentives reward keeping data, but we must change the incentives. We argue that the data are more valuable than the papers that arise from them, and so funders of research should be thinking hard about how to reward the production of high quality data. At the moment huge value is being lost from data being locked away, and data have a longer lifespan than papers. At the very least funders should be willing to meet the costs of data sharing that Tom identifies.The confidentiality risk is, I fear, exaggerated. The obvious response is for researchers to get consent from participants for data to be shared at the same time as minimising the risk of exposure. It used to be that doctors did not get consent from patients for the sharing of case reports, but now they have to—and few patients refuse. The risk of exposure from participation in a trial is way below that of a case report."
}
]
},
{
"id": "13824",
"date": "13 May 2016",
"name": "Heather M Goodare",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for asking me to comment on this paper.\n\nI can only speak from the point of view of patients and carers. The main problem is that of confidentiality of data, and some patients are worried about this. The authors acknowledge that this could be a problem (a sixth excuse). Anonymising data is of course essential, but 'small risks remain'. Remember the case of the anonymous male with back problems who was written about in an American medical journal and turned out (without too much detective work) to be President Kennedy? Personally I don't care tuppence who knows that I have had breast cancer - it's in the public domain anyway. But some conditions people would not wish to be known about: abortions, STD, some mental illnesses, and so on. If data are anonymised that is usually sufficient safeguard, but in epidemiological studies unique postcodes are a giveaway. I am a member of the Public Panel of the FARR Institute in Scotland, and we have debated the matter of Big Data at length. We have a system here called SHARE, where if you are happy for your data to be used for research you sign a form, obtainable from your GP's surgery. This also gives permission for the residue of blood samples taken for routine purposes to be used for research. Most people are happy, but some are not, even given the guarantee of anonymity. However, this system gives permission for data culled from healthcare registries to be used for research: it does not as far as I know include data from trials already conducted. This to me is a new idea, and it raises different issues. FARR talks about 'safe havens' for data, so that personal details cannot be shared and anonymity is guaranteed. It seems to me that if data already gathered for research are to be released to researchers other than the original investigators, this raises an entirely new issue. It would mean that consent forms should be revised so that they take account of the possibility that data will be shared with others at a later date. It is important to make it clear that healthcare data are not the property of the researchers who have only borrowed them: they belong to the patient. Therefore, if data are to be made more widely available, the patient needs to give consent. This means that consent forms need to make this explicit, and all other data used for research, for instance epidemiological studies that do not require active co-operation from the patient, need to have blanket consent from patients, who should be encouraged to complete a SHARE form.Personally, I like to know what researchers are going to do with my data. My husband and I were ‘consulted’ as members of a patient reference group about a stroke trial (he has had a stroke), and we both felt that it should not have gone ahead: the rest of the patient group thought so too, but it went ahead anyway. I don’t know how it got through Ethics. The relevance for this paper is that patients do have a right to say what their data are going to be used for. If they don’t approve of the trial, then they won’t let their data be used. If they have given permission initially for a study that they approve of, and the proposal is to share the data further, should they not be given a say in what their data are to be used for subsequently? Once Big Pharma get their hands on the data who knows what will become of it. This makes the sharing of data more complicated, but I believe it should be done, and that this article needs to take account of these issues.I also have some minor copy-edit suggestions: Abstract: “[…]competitors might reach different conclusions[…]” The seven incentives not to share: “[…]for employers and funders to recognise that judging researchers is core business that should not be outsourced to the arbitrary and corrupt publishing process.” The seven incentives not to share: “This cannot be in the interest of patients, who clearly want to know whether the treatment is effective or not […]” The seven incentives not to share: “There are no insurmountable technical reasons for not sharing and publishing data.”",
"responses": []
},
{
"id": "14294",
"date": "13 Jun 2016",
"name": "Gustav Nilsonne",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 piece describes and refutes seven arguments against sharing research data. The authors focus on clinical trials, but their reasoning is applicable to research with human participants in general.\nIn the ongoing conversation about open research data in scientific journals, arguments against open data are not always presented clearly and explicitly. The mere listing of counterarguments in a paper that can be referenced is therefore an important contribution.\nThe authors refute each argument against data sharing in a clear and coherent manner and their counterarguments are a valuable resource for researchers debating open data.\nI have only one minor point of criticism: the statement in the last paragraph that a study with 10 000 participants has 100 times more information content than a study with 100 participants does not take into account the diminishing information content in consecutive dependent observations. I suggest this may be reworded.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-781
|
https://f1000research.com/articles/5-780/v1
|
29 Apr 16
|
{
"type": "Review",
"title": "Irritable bowel syndrome: new insights into symptom mechanisms and advances in treatment",
"authors": [
"Robin Spiller"
],
"abstract": "Despite being one of the most common conditions leading to gastroenterological referral, irritable bowel syndrome (IBS) is poorly understood. However, recent years have seen major advances. These include new understanding of the role of both inflammation and altered microbiota as well as the impact of dietary intolerances as illuminated by magnetic resonance imaging (MRI), which has thrown new light on IBS. This article will review new data on how excessive bile acid secretion mediates diarrhea and evidence from post infectious IBS which has shown how gut inflammation can alter gut microbiota and function. Studies of patients with inflammatory bowel disease (IBD) have also shown that even when inflammation is in remission, the altered enteric nerves and abnormal microbiota can generate IBS-like symptoms. The efficacy of the low FODMAP diet as a treatment for bloating, flatulence, and abdominal discomfort has been demonstrated by randomized controlled trials. MRI studies, which can quantify intestinal volumes, have provided new insights into how FODMAPs cause symptoms. This article will focus on these areas together with recent trials of new agents, which this author believes will alter clinical practice within the foreseeable future.",
"keywords": [
"IBS",
"IBD",
"MRI studies",
"FODMAP diet",
"IBS treatment advances"
],
"content": "Introduction\n\nIrritable bowel syndrome (IBS) is one of the most common gastroenterological diagnoses, experienced by around 11% of the population. Symptoms consist of abdominal pain associated with erratic bowel habit and variable changes in stool form and frequency, suggesting considerable heterogeneity in underlying mechanisms. Despite IBS’ high prevalence, these mechanisms are poorly understood and treatment is unsatisfactory. This important unmet clinical need is a very active research area. This article will focus on recent significant advances, which this author believes are likely to influence clinical practice within the foreseeable future. These include better understanding of the role of bile acids in causing diarrhea/constipation, significance of alterations in gut microbiota, alterations in enteric innervation and serotonin availability associated with inflammation, IBS-like symptoms in patients with quiescent inflammatory bowel disease (IBD), and several new treatments including the low-FODMAP diet, guanylate cyclase C activators, and 5-hydroxytryptamine 3 (5-HT3) receptor antagonists.\n\n\nNew insights into mechanisms of IBS symptoms\n\nThe laxative properties of bile acids have been recognized since 1868, when ‘liver’ pills composed of ox bile were patented and widely promoted as panacea supplements. Scientific study had to wait until the 1970s, when perfusion studies showed that bile salts stimulated enterocyte secretion and in excess caused diarrhea. Initially, terminal ileal resection was identified as the commonest cause of bile acid diarrhea, but it is now recognized that the negative feedback loop controlling hepatic bile acid production may be disturbed in patients with intact intestines. Bile acids absorbed in the human ileum activate the nuclear receptor farnesoid X to stimulate the production of fibroblast growth factor 19 (FGF19). This circulates to the liver where, acting via the FGF receptor 4, it inhibits cholesterol 7-hydroxylase (CYP7A1), the rate-limiting enzyme that converts cholesterol to 7α-Hydroxy-4-cholesten-3-one (C4), an intermediate step in the production of cholic and chenodeoxycholic acid. Decreased circulating FGF19 leading to excessive production of bile acids can be primary or secondary to bile salt malabsorption caused by ileal resection or ileitis. If excessive bile acids enter the colon, they stimulate colonic secretion and increase stool water. A meta-analysis suggests that 10% of patients with IBS with diarrhea (IBS-D)-like symptoms have severe bile acid malabsorption, with <5% retention at 7 days1. A recent survey in the UK suggests that bile acid diarrhea accounts for nearly one in four of IBS patients referred to secondary care with diarrhea2.\n\nIdentifying patients with overproduction of bile salts used to depend on demonstrating reduced 7-day retention of an artificial radiolabeled bile acid, selenium-75 homocholic acid taurine (SeHCAT), the normal being >15%. Retention of <5% is associated with a 96% response to cholestyramine1, while lesser degrees of malabsorption produce less favorable results, with only 37% responding who have SeHCAT values >5% but <10%3. Access to SeHCAT is limited worldwide, so the recent demonstration that a fasting FGF19 <145 pg/ml predicts SeHCAT <10% with a negative predictive value of 82% and a positive predictive value of 61%4 suggests an alternative, which, as it is an enzyme-linked immunosorbent assay (ELISA), could be widely used unlike older HPLC methods. Newer, more convenient assays for C4 are also being developed. What causes a low FGF19 level in each case remains to be determined, but some cases of bile acid malabsorption begin acutely after an episode of ileitis, which is a common feature of both Salmonella species and Campylobacter jejuni gastroenteritis. Sudden onset associated with high-volume nocturnal diarrhea are characteristic features5.\n\nThe laxative effects of bile acids has been exploited by inhibitors of bile acid uptake such as elobixibat, which reduce FGF19, increase bile acid synthesis, and have been shown in phase II studies to be effective treatments for constipation6,7.\n\nThe variability in symptoms with bile acid diarrhea suggests individual differences in sensitivity to bile acids. A single nucleotide polymorphism, rs11554825, in the membrane-bound bile acid receptor TGR5 (G-protein-coupled bile acid receptor 1, also known as GpBAR1) has been suggested to be linked to small bowel and colonic transit, which were faster with TT versus TC/CC variants8. Further, more detailed studies in a smaller group showed faster colonic transit with both TT and CC TGR5 variants, possibly due to an interaction with klotho β (KLB)9. However, more work is needed as these studies are underpowered and the functional significance of the rs11554825 variants in TGR5 has yet to be established.\n\nIBD, particularly Crohn’s disease, can mimic many IBS symptoms during acute inflammatory flares, but it is increasingly recognized that acute inflammation leaves persistent changes in both nerve and muscle, which leads to IBS-like symptoms, even during remission10,11. Occult inflammation can be detected with increases in fecal calprotectin in some cases12, but that still leaves around one-third with IBS-like symptoms13. The underlying mechanisms may include altered permeability and ongoing low-level immune activation, as has been shown in the cecal biopsies of IBD patients in apparent remission but with IBS symptoms14. Other possible mechanisms include persisting alterations in enteric nerves and serotonin signaling (see below). The importance here is to recognize that such symptoms may respond better to IBS treatment including dietary restrictions rather than increasing immunosuppression with all of its inherent risks.\n\nSeveral recent studies have examined mucosal innervation in IBS and found increases in nerves expressing the transient receptor potential vanilloid channel (TRPV1)15, a peptide associated with pain pathways which also plays a key role in mechanosensitivity16. TRPV1 is upregulated by inflammation and has been shown to be increased in the rectosigmoid mucosa of IBD patients who continue to experience pain despite apparent disease quiescence17. Proximity of activated mast cells to enteric nerves has been shown to correlate with severity of abdominal pain in IBS18, and more recently a study of 101 IBS patient biopsies has shown increased amounts of neural tissue and increases in the growth-associated protein 43 (GAP43). Furthermore, biopsy supernatants increased neurogenesis in primary culture of enteric neurons19. Whether this stimulation of nerve growth causes the close association of enteric nerves and mast cells and contributes to visceral hypersensitivity in IBS remains to be determined.\n\n\nAlterations of serotonin transporter\n\nThe action of 5-HT at the synapse is terminated by active reuptake of 5-HT by the serotonin transporter (SERT). Several studies in IBS patients have suggested impairment of SERT in both platelets20 and duodenal mucosa21, though the evidence in the colon is contradictory, with some reporting a decrease22,23 and others no change24. Many such mechanistic studies use small numbers of patients, so, given the heterogeneity of IBS, conflicting results are not unexpected. The existence of subgroups of patients with abnormally increased or decreased mucosal 5-HT means that while some will respond to 5-HT receptor antagonists, others need 5-HT agonists. A polymorphism in the promoter region of the SERT gene alters SERT efficiency with the long form ll increasing efficiency and being associated with IBS with constipation (IBS-C), while the short form ss is increased in IBS-D25. Genetic differences in tryptophan hydroxylase-1 enzyme (TPH-1), the rate-limiting step in 5-HT synthesis, has been reported to predict response to 5-HT3 receptor antagonists26. Similarly, the SERT promoter polymorphism has been reported to predict response to alosetron in IBS-D with sl genotype showing reduced responsiveness27, but this was not confirmed in a larger trial with ondansetron28.\n\nMagnetic resonance imaging (MRI) provides a unique opportunity to image the undisturbed gut, since by using a range of sequences adequate contrast can be obtained with normal gut contents29. Such studies have provided for the first time accurate assessments of small bowel and regional colonic volumes in normal subjects. The resting small bowel contains surprisingly little free water, varying in different studies from 50 ml30 to 150 ml31. This rises rapidly to around 400 ml after an osmotic stress such as that provided by mannitol30 or fructose32 or falls when readily absorbable fluids are provided such as sucrose and glucose30. High-fat meals, by contrast, cause a rapid rise in small bowel water content probably due to stimulation of pancreaticobiliary secretions33.\n\nMRI has shown that there is a very wide normal range of colonic volumes with the ascending colon being mean (standard deviation [SD]) 203 (75) ml, transverse colon 232 (100) ml, and descending colon 151 (71) ml, with total colonic volumes being 632 (167) ml. While IBS-D patients have fasting colonic volumes within the normal range, those with functional constipation have significantly increased ascending colon and total colon volumes at 597 (170) and 1505 (387) ml, respectively34. Interestingly, by contrast, 95% of patients with IBS-C had colonic volumes within the normal range. In addition to fasting scans, it is simple to assess the response to feeding and also to a stronger stimulus provided by the osmotic laxative MoviprepR using cine MRI. This shows marked impairment of colonic motility in functional constipation but not IBS-C (Figure 1)34. The technique can also be used to show the mode of action of therapeutic agents including Movicol, loperamide, ondansetron, and ispaghula and could be useful in the future when screening drugs designed to alter colonic transit29.\n\n(a) Sagittal magnetic resonance image of ascending colon taken during cine recording. A system of image registration removes the movement due to diaphragmatic movements during respiration. The operator draws lines at right angles to the colonic axis and these lines are automatically propagated through the cine series. The change in line length between time points gives the transverse wall velocity and the motility index (MI) = % of lines at all time points in which the change in transverse wall velocity is >0.5 mm/s. (b) Motility index of the ascending colon following ingestion of 1L of the osmotic laxative Moviprep commencing at time -60 minutes. This shows the normal rapid increase in motility in healthy volunteers (HV) with markedly impaired response in patients with functional constipation (FC). Irritable bowel syndrome with constipation (IBS-C) patients showed an initially normal response which had faded by the second hour. Data from Lam et al.34.\n\nMany IBS patients report that their symptoms are aggravated by eating certain foods, and several uncontrolled studies found 36–40% of patients could be helped by selective exclusion of a range of foods often including dairy, wheat, onions, and fruit35,36. Double blind exclusion diets are very demanding and few have been done until recently, when a clearer definition of what was being excluded was developed based on the FODMAP concept (Text Box 1).\n\nSystematic application of this diet required the chemical analysis of common foods to identify those that contained significant amounts of these substances. The pioneers were the group in Monash University led by Gibson, a gastroenterologist, and Muir, a biochemist. The most important sources of FODMAPs in the western diet are wheat, onions, and fruit with an excess of fructose over glucose such as apples and pears. Dairy products are also important in those with lactose malabsorption. The first rigorous placebo-controlled diet published in 2014 showed that the low-FODMAP diet improved bloating and abdominal pain/discomfort when compared to the standard Australian diet37. It should be noted, however, that the low-FODMAP diet does not alter bowel habit consistently and so cannot be expected to benefit those whose main problem is diarrhea or constipation. The low-FODMAP diet has been compared with an ‘IBS diet’ in a randomized trial that showed a similar improvement in symptoms38. There is some overlap between the two diets, but the ‘IBS diet’ gives more general advice like regular meals and exercise along with avoiding foods thought to promote gas formation without the rigor of a diet based on the chemical composition of food. The complexity of the FODMAP diet makes it difficult to implement, an obstacle which perhaps could be overcome by excluding just the major sources of FODMAPs in any individual’s diet (e.g. wheat, onions, and dairy) and not bothering with items which individually contribute only small amounts or dairy products in those with the lactose persistence genotype39.\n\nThe advances associated with non-culture-based methods of microbiota assessment using ribosomal RNA analysis has led to numerous studies of the microbiota in IBS with somewhat conflicting results (for review, see 40). This is perhaps not surprising, since it is clear that diet and transit are both major determinants of microbiota composition and most studies of the microbiota in IBS have failed to control for these factors41. Fast transit, such as is seen in IBS-D patients, gives a survival advantage to either organisms that multiply rapidly or those that adhere well to the mucosa42. This latter study showed that stool consistency was an important predictor of enterotype but did not comment on how consistent this was within an individual. One of the key features of IBS is the erratic pattern of stool form43, with both hard and loose stool within a time period as short as 24 hours, suggesting that stool microbiota might also be unstable in IBS. A useful study demonstrated that while a subgroup have microbiota that are distinctly different from normal, many IBS patients have microbiota that cluster with normal controls. Interestingly, the group with ‘normal’ microbiota were more likely to have clinically significant depression, suggesting that abnormal microbiota might represent a group with a predominantly peripheral gut abnormality, while normal microbiota might be a signature of those in whom the main abnormality lies centrally44. Acute gastroenteritis causes a marked disturbance of the gut microbiota with overgrowth of pathogen and a marked reduction in diversity. This is followed by a return towards the former equilibrium but, as recently shown, distinct differences remain after Campylobacter jejuni enteritis, the commonest cause of food poisoning in the UK. Restricting analysis to just 27 genus-like taxa, we showed in a redundancy analysis that the individuals could be ordered on the primary axis from health at one end to IBS at the other with infected but recovered individuals in between45 (Figure 2).\n\nThe primary axis was used as an index of dysbiosis, which separates these groups in a graded fashion from health to disease. Reproduced from Jalanka et al.45.\n\nThis is an area of considerable confusion, largely because there is a gradient of bacterial density within the small bowel ranging from 10-1 ml in the duodenum to 107 ml in the terminal ileum. Identifying an abnormal increase in luminal bacteria requires defining this gradient throughout the small bowel, something which current tests cannot do. Intubation and analysis of aspirates give a single value, usually from the jejunum. Using such a test, values exceeding the accepted threshold of 105/ml are found in around 4% of both controls and IBS patients46. Lactulose breath hydrogen tests (LBHTs) alone are impossible to interpret, as one can rarely exclude the possibility that any early rise in breath hydrogen observed is due to lactulose reaching the cecum. However, this can, to some extent, be overcome by combining with scintigraphic assessment of orocecal transit (SOCT), which gives a time when >5% of isotope has entered the colon. If the breath hydrogen rise occurs before this time then it is usually interpreted as showing abnormal microbiota in the small bowel. However, it should be noted that this requires that breath hydrogen would not rise if <5%, i.e. 0.5 g, had entered the colon, which is an unproven assumption, as this amount of lactulose will produce 16 ml of hydrogen47, which, depending on the rate of excretion, could raise breath hydrogen by much more than the 10 parts per million required for a positive test. The terminal ileum is an area where the stasis which favors growth of colonic anaerobes is often observed, especially during fasting48, so this is most likely to be the site where microbiota would proliferate. A recent study using this technique suggested that around one-third of IBS patients attending a Chinese outpatient clinic have a positive LBHT/SOCT, though it is unclear how these were selected or whether this finding is generalizable to other clinics. A positive test did seem to predict a better response to rifaximin, but these studies need repeating as the numbers were very small49. These values are comparable with a previous large study using the glucose breath hydrogen test, which reported positive tests in 31% of 65 IBS patients versus 4% in 105 healthy controls50.\n\nWhere the history is confusing, colonic transit may be helpful in predicting response to drugs accelerating or retarding transit51. Low SeHCAT values predict response to cholestyramine, but otherwise there are currently few other biomarkers in clinical use that can predict response to specific therapies. Measures of visceral sensitivity do not appear to predict response to ketanserin, even though this did increase the threshold for discomfort during a barostat study52. Genetic tests might fare better, but preliminary reports suggesting TPH-1 polymorphisms predict response to ramosetron need confirmation in larger studies26. Genetic testing for lactose intolerance could replace LBHT, being more convenient and highly sensitive53. Though interesting mechanistically, the mucosal biopsy assays including histology and mediator release show such wide variability that none so far are useful diagnostically nor in predicting treatment response.\n\n\nAdvances in treatment for IBS\n\nLoperamide is a safe and effective anti-diarrheal agent, which has been shown in randomized controlled trials (RCTs) to reduce bowel frequency in IBS-D but with little benefit on pain54, which may actually increase55. Despite this, loperamide improves quality of life since it allows planning of trips and socializing, which anxious IBS-D patients often avoid for fear of fecal urgency or even incontinence. More recently, eluxadoline, a mu-opioid receptor agonist with delta-opioid receptor antagonistic action, has been shown in a large, high-quality phase IIb dose-finding RCT to benefit IBS-D with a 14% increase in responder rates (28% versus 14%) after 12 weeks of either 100 or 200 mg twice daily56. However, this mode of action does carry a risk of causing acute pancreatitis through sphincter of Oddi spasm, which may prove unacceptable in IBS.\n\n5-HT3 receptor antagonists (5-HT3RAs) are effective treatments for IBS-D57, slowing transit, reducing bowel frequency, normalizing stool consistency, and reducing urgency58, which is one of the key symptoms that impair quality of life in IBS-D. Alosetron has been shown to significantly improve quality of life59. Constipation is a common side effect, which was reported in around 25% of those given standard doses of alosetron. While this can be controlled by dose reduction, this is not true of ischemic colitis, a much rarer side effect, which was seen in around one in 700 patients60,61. Although not life threatening, ischemic colitis led to alosetron’s withdrawal from general marketing, though now it is off patent, use may increase. Ramosetron is effective at a very low dose, 5 µg in men62,63 and 2.5 µg in women64, with an acceptably low rate of constipation and no reports of ischemic colitis. Recently, the much less potent 5-HT3RA ondansetron, given at a dose of 4 mg, range 2–6 mg, was shown to be highly effective at improving stool consistency (Figure 3), reducing stool frequency and reducing urgency, with 70% reporting adequate relief of IBS symptoms on ondansetron compared to 16% on placebo, giving a number needed to treat (NNT) of two28. It is worth noting that ondansetron has been used for over two decades with no reports of ischemic colitis and has an excellent safety record, a feature which is so important for IBS medication.\n\nStool form score fell into the normal range 3–5 within 1 week of starting ondansetron, rapidly returning to baseline on discontinuation. There was very little placebo response. Reproduced from Garsed et al.28.\n\nRecently, two new secretagogues, lubiprostone and linaclotide, have been introduced for the treatment of both chronic constipation and IBS-C. Both are supported by large RCTs performed to high standards65–68. Lubiprostone stimulates chloride secretion by activating the type 2 chloride ion channel69. It does not alter pain thresholds during rectal distension using a barostat70 but does accelerate transit in healthy volunteers71. It relieves overall IBS-C symptoms with 17.9% responder rate compared to 10.1% with placebo and this is associated with modest reductions in pain and straining65. This gives a NNT of 13. Around 25% of patients experience nausea, but only around 5% discontinue its use because of this. Linaclotide also stimulates chloride secretion by activating the guanylate cyclase C receptor, increasing cyclic guanosine monophosphate (cGMP), an important intracellular second messenger that activates the cystic fibrosis transmembrane conductance regulator (CFTR) to cause chloride secretion72. Linaclotide, 290 µg daily, improved both pain and stool consistency in a 12-week trial of 800 IBS-C patients, meeting the FDA guidelines in 33.6% compared to 21% on placebo, giving an NNT of eight73. The benefit persisted in another 26-week trial67. Both trials showed improvement in stool consistency and frequency within the first week, while abdominal discomfort continued to improve over a period of about 6 weeks. The main side effect is diarrhea, experienced in 19.7%, severe in 2%, and leading to drug discontinuation in 4.5%6.\n\n\nFuture directions\n\nThe heterogeneity of IBS means that future studies must aim to be larger and more mechanistic than in the past. There are too many small studies performed by isolated research groups with contradictory conclusions. These will be resolved only by larger multicenter studies involving consortia of researchers from many different areas. Such studies should include not only symptom responses but also measurement of relevant biomarkers, which will confirm or refute potential mechanisms. Our current symptom-based criteria for study entry led to small differences from placebo in therapeutic trials because similar symptoms can arise from more than one mechanism. We should subdivide our patients by the mechanisms discussed above and use as entry criteria to trials so that therapies are targeted to those who can benefit, with a potential for reducing the time and cost of developing new effective therapies. Better biomarkers more directly assessing the disturbance in function being targeted will help in this process.",
"appendix": "Competing interests\n\n\n\nRobin Spiller has received research funding from Lesaffre and Ironwood. He has also acted on advisory boards for Almirall, Yuhan, Ipsen, Commonwealth Diagnostics International and Danone and received speakers’ fees from Menarini.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nWedlake L, A'Hern R, Russell D, et al.: Systematic review: the prevalence of idiopathic bile acid malabsorption as diagnosed by SeHCAT scanning in patients with diarrhoea-predominant irritable bowel syndrome. Aliment Pharmacol Ther. 2009; 30(7): 707–717. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAziz I, Mumtaz S, Bholah H, et al.: High Prevalence of Idiopathic Bile Acid Diarrhea Among Patients With Diarrhea-predominant Irritable Bowel Syndrome Based on Rome III Criteria. Clin Gastroenterol Hepatol. 2015; 13(9): 1650–5.e2. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWilliams AJ, Merrick MV, Eastwood MA: Idiopathic bile acid malabsorption--a review of clinical presentation, diagnosis, and response to treatment. Gut. 1991; 32(9): 1004–1006. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPattni SS, Brydon WG, Dew T, et al.: Fibroblast growth factor 19 in patients with bile acid diarrhoea: a prospective comparison of FGF19 serum assay and SeHCAT retention. Aliment Pharmacol Ther. 2013; 38(8): 967–976. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSinha L, Liston R, Testa HJ, et al.: Idiopathic bile acid malabsorption: qualitative and quantitative clinical features and response to cholestyramine. Aliment Pharmacol Ther. 1998; 12(9): 839–844. 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PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCamilleri M, Shin A, Busciglio I, et al.: Genetic variation in GPBAR1 predisposes to quantitative changes in colonic transit and bile acid excretion. Am J Physiol Gastrointest Liver Physiol. 2014; 307(5): G508–G516. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHalpin SJ, Ford AC: Prevalence of symptoms meeting criteria for irritable bowel syndrome in inflammatory bowel disease: systematic review and meta-analysis. Am J Gastroenterol. 2012; 107(10): 1474–1482. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSpiller R, Lam C: The shifting interface between IBS and IBD. Curr Opin Pharmacol. 2011; 11(6): 586–592. PubMed Abstract | Publisher Full Text\n\nKeohane J, O'Mahony C, O'Mahony L, et al.: Irritable bowel syndrome-type symptoms in patients with inflammatory bowel disease: a real association or reflection of occult inflammation? Am J Gastroenterol. 2010; 105(8): 1788, 1789–94; quiz 1795. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBerrill JW, Green JT, Hood K, et al.: Symptoms of irritable bowel syndrome in patients with inflammatory bowel disease: examining the role of sub-clinical inflammation and the impact on clinical assessment of disease activity. Aliment Pharmacol Ther. 2013; 38(1): 44–51. PubMed Abstract | Publisher Full Text\n\nVivinus-Nébot M, Frin-Mathy G, Bzioueche H, et al.: Functional bowel symptoms in quiescent inflammatory bowel diseases: role of epithelial barrier disruption and low-grade inflammation. Gut. 2014; 63(5): 744–752. 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PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBarbara G, Stanghellini V, De Giorgio R, et al.: Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome. Gastroenterology. 2004; 126(3): 693–702. PubMed Abstract | Publisher Full Text\n\nDothel G, Barbaro MR, Boudin H, et al.: Nerve fiber outgrowth is increased in the intestinal mucosa of patients with irritable bowel syndrome. Gastroenterology. 2015; 148(5): 1002–1011.e4. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBellini M, Rappelli L, Blandizzi C, et al.: Platelet serotonin transporter in patients with diarrhea-predominant irritable bowel syndrome both before and after treatment with alosetron. Am J Gastroenterol. 2003; 98(12): 2705–2711. PubMed Abstract | Publisher Full Text\n\nFoley S, Garsed K, Singh G, et al.: Impaired uptake of serotonin by platelets from patients with irritable bowel syndrome correlates with duodenal immune activation. Gastroenterology. 2011; 140(5): 1434–43.e1. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nCoates MD, Mahoney CR, Linden DR, et al.: Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome. Gastroenterology. 2004; 126(7): 1657–1664. PubMed Abstract | Publisher Full Text\n\nKerckhoffs AP, ter Linde JJ, Akkermans LM, et al.: SERT and TPH-1 mRNA expression are reduced in irritable bowel syndrome patients regardless of visceral sensitivity state in large intestine. Am J Physiol Gastrointest Liver Physiol. 2012; 302(9): G1053–G1060. 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PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCamilleri M, Atanasova E, Carlson PJ, et al.: Serotonin-transporter polymorphism pharmacogenetics in diarrhea-predominant irritable bowel syndrome. Gastroenterology. 2002; 123(2): 425–432. PubMed Abstract | Publisher Full Text\n\nGarsed K, Chernova J, Hastings M, et al.: A randomised trial of ondansetron for the treatment of irritable bowel syndrome with diarrhoea. Gut. 2014; 63(10): 1617–1625. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKhalaf A, Hoad CL, Spiller RC, et al.: Magnetic resonance imaging biomarkers of gastrointestinal motor function and fluid distribution. World J Gastrointest Pathophysiol. 2015; 6(4): 140–149. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarciani L, Cox EF, Hoad CL, et al.: Postprandial changes in small bowel water content in healthy subjects and patients with irritable bowel syndrome. Gastroenterology. 2010; 138(2): 469–77, 477.e1. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMarciani L, Wright J, Foley S, et al.: Effects of a 5-HT3 antagonist, ondansetron, on fasting and postprandial small bowel water content assessed by magnetic resonance imaging. Aliment Pharmacol Ther. 2010; 32(5): 655–663. PubMed Abstract | Publisher Full Text\n\nMurray K, Wilkinson-Smith V, Hoad C, et al.: Differential effects of FODMAPs (fermentable oligo-, di-, mono-saccharides and polyols) on small and large intestinal contents in healthy subjects shown by MRI. Am J Gastroenterol. 2014; 109(1): 110–119. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHussein MO, Hoad CL, Wright J, et al.: Fat emulsion intragastric stability and droplet size modulate gastrointestinal responses and subsequent food intake in young adults. J Nutr. 2015; 145(6): 1170–1177. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLam C, Chaddock G, Marciani L, et al.: Colonic response to laxative ingestion as assessed by MRI differs in constipated irritable bowel syndrome compared to functional constipation. Neurogastroenterol Motil. 2016. PubMed Abstract | Publisher Full Text\n\nNanda R, James R, Smith H, et al.: Food intolerance and the irritable bowel syndrome. Gut. 1989; 30(8): 1099–1104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParker TJ, Naylor SJ, Riordan AM, et al.: Management of patients with food intolerance in irritable bowel syndrome: The development and use of an exclusion diet. J Human Nutrition Dietetics. 1995; 8(3): 159–166. Publisher Full Text\n\nHalmos EP, Power VA, Shepherd SJ, et al.: A diet low in FODMAPs reduces symptoms of irritable bowel syndrome. Gastroenterology. 2014; 146(1): 67–75.e5. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBohn L, Storsrud S, Liljebo T, et al.: Diet low in FODMAPs reduces symptoms of irritable bowel syndrome as well as traditional dietary advice: a randomized controlled trial. Gastroenterology. 2015; 149(6): 1399–1407.e2. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPohl D, Savarino E, Hersberger M, et al.: Excellent agreement between genetic and hydrogen breath tests for lactase deficiency and the role of extended symptom assessment. Br J Nutr. 2010; 104(6): 900–907. PubMed Abstract | Publisher Full Text\n\nSimrén M, Barbara G, Flint HJ, et al.: Intestinal microbiota in functional bowel disorders: a Rome foundation report. Gut. 2013; 62(1): 159–176. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRajilic-Stojanovic M, Jonkers DM, Salonen A, et al.: Intestinal microbiota and diet in IBS: causes, consequences, or epiphenomena? Am J Gastroenterol. 2015; 110(2): 278–287. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVandeputte D, Falony G, Vieira-Silva S, et al.: Stool consistency is strongly associated with gut microbiota richness and composition, enterotypes and bacterial growth rates. Gut. 2016; 65(1): 57–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPimentel M, Hwang L, Melmed GY, et al.: New clinical method for distinguishing D-IBS from other gastrointestinal conditions causing diarrhea: the LA/IBS diagnostic strategy. Dig Dis Sci. 2010; 55(1): 145–149. PubMed Abstract | Publisher Full Text\n\nJeffery IB, O'Toole PW, Ohman L, et al.: An irritable bowel syndrome subtype defined by species-specific alterations in faecal microbiota. Gut. 2012; 61(7): 997–1006. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nJalanka-Tuovinen J, Salojarvi J, Salonen A, et al.: Faecal microbiota composition and host-microbe cross-talk following gastroenteritis and in postinfectious irritable bowel syndrome. Gut. 2014; 63(11): 1737–1745. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPosserud I, Stotzer PO, Bjornsson ES, et al.: Small intestinal bacterial overgrowth in patients with irritable bowel syndrome. Gut. 2007; 56(6): 802–808. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nChristl SU, Murgatroyd PR, Gibson GR, et al.: Production, metabolism, and excretion of hydrogen in the large intestine. Gastroenterology. 1992; 102(4 Pt 1): 1269–1277. PubMed Abstract\n\nSpiller RC, Brown ML, Phillips SF: Emptying of the terminal ileum in intact humans. Influence of meal residue and ileal motility. Gastroenterology. 1987; 92(3): 724–729. PubMed Abstract\n\nZhao J, Zheng X, Chu H, et al.: A study of the methodological and clinical validity of the combined lactulose hydrogen breath test with scintigraphic oro-cecal transit test for diagnosing small intestinal bacterial overgrowth in IBS patients. Neurogastroenterol Motil. 2014; 26(6): 794–802. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLupascu A, Gabrielli M, Lauritano EC, et al.: Hydrogen glucose breath test to detect small intestinal bacterial overgrowth: a prevalence case-control study in irritable bowel syndrome. Aliment Pharmacol Ther. 2005; 22(11–12): 1157–1160. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSpiller RC: Potential biomarkers. Gastroenterol Clin North Am. In: Chey WD, editor. Irritable bowel syndrome. 40 ed. Elsevier; 2011; 40(1): 121–139. PubMed Abstract | Publisher Full Text\n\nKlooker TK, Braak B, Koopman KE, et al.: The mast cell stabiliser ketotifen decreases visceral hypersensitivity and improves intestinal symptoms in patients with irritable bowel syndrome. Gut. 2010; 59(9): 1213–1221. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKrawczyk M, Wolska M, Schwartz S, et al.: Concordance of genetic and breath tests for lactose intolerance in a tertiary referral centre. J Gastrointestin Liver Dis. 2008; 17(2): 135–139. PubMed Abstract\n\nLavö B, Stenstam M, Nielsen AL: Loperamide in treatment of irritable bowel syndrome--a double-blind placebo controlled study. Scand J Gastroenterol Suppl. 1987; 130: 77–80. PubMed Abstract | Publisher Full Text\n\nEfskind PS, Bernklev T, Vatn MH: A double-blind placebo-controlled trial with loperamide in irritable bowel syndrome. Scand J Gastroenterol. 1996; 31(5): 463–468. PubMed Abstract | Publisher Full Text\n\nDove LS, Lembo A, Randall CW, et al.: Eluxadoline benefits patients with irritable bowel syndrome with diarrhea in a phase 2 study. Gastroenterology. 2013; 145(2): 329–338.e1. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAndresen V, Montori VM, Keller J, et al.: Effects of 5-hydroxytryptamine (serotonin) type 3 antagonists on symptom relief and constipation in nonconstipated irritable bowel syndrome: a systematic review and meta-analysis of randomized controlled trials. Clin Gastroenterol Hepatol. 2008; 6(5): 545–555. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLembo AJ, Olden KW, Ameen VZ, et al.: Effect of alosetron on bowel urgency and global symptoms in women with severe, diarrhea-predominant irritable bowel syndrome: analysis of two controlled trials. Clin Gastroenterol Hepatol. 2004; 2(8): 675–682. PubMed Abstract | Publisher Full Text\n\nCremonini F, Nicandro JP, Atkinson V, et al.: Randomised clinical trial: alosetron improves quality of life and reduces restriction of daily activities in women with severe diarrhoea-predominant IBS. Aliment Pharmacol Ther. 2012; 36(5): 437–448. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nChang L, Chey WD, Harris L, et al.: Incidence of ischemic colitis and serious complications of constipation among patients using alosetron: systematic review of clinical trials and post-marketing surveillance data. Am J Gastroenterol. 2006; 101(5): 1069–1079. PubMed Abstract | Publisher Full Text\n\nChang L, Tong K, Ameen V: Ischemic colitis and complications of constipation associated with the use of alosetron under a risk management plan: clinical characteristics, outcomes, and incidences. Am J Gastroenterol. 2010; 105(4): 866–875. PubMed Abstract | Publisher Full Text\n\nChiba T, Yamamoto K, Sato S: Long-term efficacy and safety of ramosetron in the treatment of diarrhea-predominant irritable bowel syndrome. Clin Exp Gastroenterol. 2013; 6: 123–128. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMatsueda K, Harasawa S, Hongo M, et al.: A randomized, double-blind, placebo-controlled clinical trial of the effectiveness of the novel serotonin type 3 receptor antagonist ramosetron in both male and female Japanese patients with diarrhea-predominant irritable bowel syndrome. Scand J Gastroenterol. 2008; 43(10): 1202–1211. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFukudo S, Kinoshita Y, Okumura T, et al.: Ramosetron Reduces Symptoms of Irritable Bowel Syndrome With Diarrhea and Improves Quality of Life in Women. Gastroenterology. 2016; 150(2): 358–366.e8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDrossman DA, Chey WD, Johanson JF, et al.: Clinical trial: lubiprostone in patients with constipation-associated irritable bowel syndrome--results of two randomized, placebo-controlled studies. Aliment Pharmacol Ther. 2009; 29(3): 329–341. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nChey WD, Drossman DA, Johanson JF, et al.: Safety and patient outcomes with lubiprostone for up to 52 weeks in patients with irritable bowel syndrome with constipation. Aliment Pharmacol Ther. 2012; 35(5): 587–599. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nChey WD, Lembo AJ, Lavins BJ, et al.: Linaclotide for irritable bowel syndrome with constipation: a 26-week, randomized, double-blind, placebo-controlled trial to evaluate efficacy and safety. Am J Gastroenterol. 2012; 107(11): 1702–1712. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLembo AJ, Schneier HA, Shiff SJ, et al.: Two randomized trials of linaclotide for chronic constipation. N Engl J Med. 2011; 365(6): 527–536. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRivkin A, Chagan L: Lubiprostone: chloride channel activator for chronic constipation. Clin Ther. 2006; 28(12): 2008–2021. PubMed Abstract | Publisher Full Text\n\nWhitehead WE, Palsson OS, Gangarosa L, et al.: Lubiprostone does not influence visceral pain thresholds in patients with irritable bowel syndrome. Neurogastroenterol Motil. 2011; 23(10): 944–e400. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCamilleri M, Bharucha AE, Ueno R, et al.: Effect of a selective chloride channel activator, lubiprostone, on gastrointestinal transit, gastric sensory, and motor functions in healthy volunteers. Am J Physiol Gastrointest Liver Physiol. 2006; 290(5): G942–G947. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBusby RW, Kessler MM, Bartolini WP, et al.: Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation. J Pharmacol Exp Ther. 2013; 344(1): 196–206. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRao S, Lembo AJ, Shiff SJ, et al.: A 12-week, randomized, controlled trial with a 4-week randomized withdrawal period to evaluate the efficacy and safety of linaclotide in irritable bowel syndrome with constipation. Am J Gastroenterol. 2012; 107(11): 1714–1724; quiz p.1725. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "13647",
"date": "29 Apr 2016",
"name": "Anton Emmanuel",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13648",
"date": "29 Apr 2016",
"name": "William Chey",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-780
|
https://f1000research.com/articles/5-776/v1
|
28 Apr 16
|
{
"type": "Review",
"title": "Kin and multilevel selection in social evolution: a never-ending controversy?",
"authors": [
"Jos Kramer",
"Joël Meunier",
"Joël Meunier"
],
"abstract": "Kin selection and multilevel selection are two major frameworks in evolutionary biology that aim at explaining the evolution of social behaviors. However, the relationship between these two theories has been plagued by controversy for almost half a century and debates about their relevance and usefulness in explaining social evolution seem to rekindle at regular intervals. Here, we first provide a concise introduction into the kin selection and multilevel selection theories and shed light onto the roots of the controversy surrounding them. We then review two major aspects of the current debate: the presumed formal equivalency of the two theories and the question whether group selection can lead to group adaptation. We conclude by arguing that the two theories can offer complementary approaches to the study of social evolution: kin selection approaches usually focus on the identification of optimal phenotypes and thus on the endresult of a selection process, whereas multilevel selection approaches focus on the ongoing selection process itself. The two theories thus provide different perspectives that might be fruitfully combined to promote our understanding of the evolution in group-structured populations.",
"keywords": [
"cooperation",
"altruism",
"sociobiology",
"group selection",
"levels of selection",
"inclusive fitness"
],
"content": "Introduction\n\nWhy should an individual cooperate to benefit others? This question pinpoints one of the central theoretical problems of sociobiology1–3. Cooperative behaviors such as altruism (an action that benefits others at one’s own expense) would reduce the fitness of the performer relative to selfish individuals that do not perform the behavior, and hence should be selected against4–6. However, this expectation is in striking contrast to the ubiquity of cooperation in nature, which occurs among ‘simple’ microorganisms7,8 and within highly complex eusocial societies alike9.\n\nKinship and group selection are two key concepts of modern sociobiology that have been proposed to help resolve this apparent conundrum1,6. Despite their common origin in the writings of Charles Darwin (cf. 10), the developments of these two concepts in the modern kin and multilevel (or group) selection theories followed diverging paths and fueled a persisting and often heated debate about their relevance and usefulness in the study of social evolution (e.g. 11–13). Here, we provide a concise introduction to the two theories and the controversy surrounding them as well as highlight the complementarity of the approaches typically taken by their proponents. To this end, we first separately introduce the two theories and then point to the roots of the controversy. We subsequently review two important aspects of the current debate in more detail: the presumed formal equivalency of the two theories and the notion of group adaptation. We overall suggest that these issues illustrate the complementary nature of the perspectives offered by the kin selection and multilevel selection theories.\n\n\nKin and multilevel selection theories in a nutshell\n\nInteracting organisms may have an evolutionary incentive to help each other (or at least to hurt each other less) if they share genes, and the magnitude of this incentive should increase with the degree of relatedness between them; this is the central tenet of William D. Hamilton’s inclusive fitness theory14–16 (the term kin selection theory was coined by John Maynard-Smith11 and is here used as a synonym for ‘inclusive fitness theory’ to comply with its conventional use). This tenet is encapsulated in a very simple form in Hamilton’s rule, which states that a (gene for a) social behavior is favored by natural selection if rb-c > 0, where c is the fitness cost to the individual performing the behavior, b equals the fitness benefit to the recipient(s), and r is the genetic relatedness between them14,15. The rule thus formalizes the realization that natural selection acts not only through direct effects of a behavior on the actor’s own fitness (often measured as reproductive output) but also through indirect effects on the fitness of the actor’s relatives (that have an above-average probability of sharing the actor’s genes, including the one[s] that cause the social behavior in question)1. Moreover, it provides a potential solution to the central problem of sociobiology, as it shows that even costly social behaviors can be favored by natural selection as long as the direct costs are outweighed by a sufficient amount of indirect benefit to sufficiently closely related individuals (Figure 1)17. Note, however, that the application of Hamilton’s rule—and thus kin selection theory—is not restricted to altruistic behaviors: rb and -c represent, respectively, the indirect and the direct fitness consequences of any character of interest and hence can both be positive, negative, or zero18. Accordingly, they can also represent mutually beneficial (both fitness components positive), spiteful (both components negative), or selfish (direct component positive and indirect component zero or negative) behaviors.\n\nIn this example, non-altruists and altruists (symbolized by triangles and squares, respectively; see the legend for details) form (I) pairs of non-altruists, (II) mixed pairs, and (III) pairs of altruists. Altruists and non-altruists do not share the gene(s) causing the altruistic behavior but are overall related (i.e. genetically similar with respect to other traits). Upon reproduction, the (F1) progeny of each pair forms an independent group. Non-altruists do not express social behaviors and reproduce according to a baseline fitness (here arbitrarily fixed at z = 3). In contrast, altruists (unconditionally) confer a fitness benefit (b = 4) onto their partner at a cost (c = 1) to themselves. Although altruists incur a direct fitness cost, they benefit indirectly from assisting their partner and hence overall increase their inclusive fitness (see the fitness box for an illustration: inclusive fitness is composed of a direct [unicolored symbols] and an indirect [symbols framed in the same color] component). Similarly, their neighbor-modulated fitness is increased if they are assisted by their partner. However, altruists have a relative fitness disadvantage within mixed pairs because they increase the fitness of their non-altruistic partner at a cost to themselves and without receiving help in return (see pay-offs box for details). Hence, altruism can be disadvantageous even within groups of overall related individuals (precisely if they do not share the altruistic gene). In contrast, uniform pairs of altruists produce more offspring than mixed groups or groups of non-altruists (they hence have a higher group fitness in an MLS-1 framework; the group fitness in an MLS-2 framework corresponds to the number of groups produced and is here the same for all pairs). Positive assortment of altruists (e.g. due to limited dispersal) reinforces this ‘group-advantageous’ effect and can explain the evolution of cooperation in the long run.\n\nAlongside Hamilton’s rule, the concept of inclusive fitness is the second central element of kin selection theory. An organism’s inclusive fitness is defined as the sum of its direct (Darwinian) and indirect fitness components (Figure 1). The latter is calculated as the relatedness-weighed sum of those effects on the fitness of other individuals for which the organism is causally responsible14,17. Inclusive fitness is thus an actor-centric approach that examines how a focal individual influences its own fitness and that of its social partners19. Hamilton14 also suggested an alternative and (by now) increasingly used approach to account for direct and indirect fitness effects (e.g. 20–22): the neighbor-modulated fitness (sometimes referred to as personal or direct fitness). The two approaches differ in how the indirect component is conceptualized: in contrast to inclusive fitness, neighbor-modulated fitness is a recipient-centric approach and thus examines how social partners influence the fitness of a focal individual19 (Figure 1). The two approaches are usually seen as equivalent, as they predict the same overall response to natural selection18 (but cf. 23,24). The inclusive fitness approach, however, comes with one significant conceptual advantage: in principle, an individual is causally responsible for both its direct and indirect fitness and hence can control its inclusive fitness. Thus, natural selection might favor organisms that act as if they are attempting to maximize their inclusive fitness14,25–27 (but cf. 28,29). The possibility of conceptualizing individuals as maximizing agents (an optimality approach30) greatly facilitates the linking of theoretical and empirical research and has been central to the study of adaptation in behavioral and evolutionary ecology19.\n\nOver the last five decades, kin selection theory has been extensively developed and generalized (e.g. 20,31–35; see 22,23 for book-length treatments) beyond the limited scope of Hamilton’s original formalization14–16. For instance, Hamilton originally defined relatedness as a genealogical measure of shared ancestry14 but quickly realized that this is only one (albeit by far the most frequent) way of generating the above-average genetic similarity among individuals that ultimately drives the evolution of many cooperative behaviors16,31,36. In contemporary discussions, relatedness is accordingly more broadly defined (e.g. 23,24) and encompasses any genetic similarity, regardless of whether it arose by common descent or by other means such as green-beard effects37,38. Moreover, the generalization of kin selection theory resulted in the development of general versions of Hamilton’s rule (most notably on the basis of the Price equation; see below and 34) that—unlike the original version—make no assumptions such as weak selection or additivity of fitness payoffs (see also 18,39,40). In these general versions, the cost-benefit parameters no longer denote simple fitness payoffs of social interactions but rather partial regression coefficients that quantify the overall statistical association among an organism’s phenotype/genotype, its fitness, and the phenotype/genotype of its social partners17,41. Concomitant with its generalization, kin selection theory has become the dominating framework for the explanation of social behavior from an evolutionary viewpoint (e.g. 42,43). Its most prominent empirical prediction, namely that social behavior should correlate with relatedness, has been supported across diverse taxa (the equally important impact of the costs and benefits received less attention, but see 44,45), and kin selection theory has greatly contributed to our current understanding of a variety of biological phenomena such as dispersal, reproductive skew, and queen-worker conflicts in eusocial insects46,47.\n\nThe central tenet of multilevel (or group) selection theory conveys that selection not only acts on individuals but can act (simultaneously) on multiple levels of biological organization, including cells and/or groups48. This view suggests that even if behaviors that benefit other individuals are selectively disadvantageous at the level of the individual, they might still evolve if they are advantageous at—and hence selected for on—a higher level of the biological hierarchy (e.g. on the group or colony level)6,48. Altruism, for instance, is costly for the altruistic individual, but groups containing a higher proportion of altruistic individuals usually have a competitive advantage over groups that are composed mostly of selfish individuals (e.g. because altruistic groups are more productive or superior in direct confrontations). In such situations, altruism can evolve—driven by a process of selection between groups—even against the background of selection favoring selfishness within each group (e.g. 49,50). This is the potential solution of the central problem of sociobiology from a multilevel perspective (Figure 1). The applicability of multilevel selection theory, however, is not limited to situations in which selection pressures on different hierarchical levels are opposing. Multilevel selection approaches more generally examine the direction and strength of (naturally occurring or experimentally applied) selection pressures on multiple hierarchical levels, investigate the mediators (e.g. indirect genetic effects51–53) and magnitude of their respective contributions to total evolutionary change50,54,55, and explore effects of selection on group traits56.\n\nInterestingly, there is currently no unanimously accepted formal theory of multilevel selection (e.g. 57–60) apart from the broad consent that defines group selection as natural selection based on the differential survival and reproduction of groups6,51,61 (but cf. 54). As a result, both individuals and groups can be found as focal units of attention (Figure 1) in multilevel selection approaches. Similarly, the definitions of group fitness and group vary among studies depending on whether they aimed at explaining the changing frequency of different types of organisms in a group-structured population or at explaining the changing frequency of different types of group in a meta-population of groups. In the first case, group fitness is often defined as the average (or total) fitness of its constituent organism (multilevel-selection-1, or MLS-1; Figure 162–64), and the group as a set of interacting individuals that influence each other’s fitness, but not the fitness of individuals outside the group, with respect to a particular trait (trait-groups49,61; see 48 for a discussion of this concept). In the second case, group fitness is defined as the expected number of offspring groups (MLS-2; individual fitness is then usually defined separately and on a different timescale62–64). MLS-2 approaches often explicitly incorporate group-level events such as fission or extinction and thus assume that groups of individuals undergoing such group-level events can be identified in the population64. Groups are consequently more narrowly defined as geographically discrete, multigenerational, and reproductively isolated demes (Figure 1)48.\n\nIt is important to note that MLS-1 and MLS-2 approaches are not equivalent, as they relate to different natural processes48 (but cf. 58). In an MLS-1 scenario, groups ‘merely’ generate the population structure that affects the fitness of individuals. Hence, groups can propagate by producing individuals as long as these individuals form groups themselves at some stage of their life (e.g. after blending in a common mating pool). In contrast, groups need to reproduce in an ordinary sense in an MLS-2 scenario (i.e. by producing more groups)48. Though sometimes seen as fostering confusion (e.g. 58,65), the difference between MLS-1 and MLS-2 also provides an intriguing diachronic perspective on ‘major transitions’ in evolution—and thus on the evolution of the biological hierarchy itself—as such transitions involve a temporal shift from MLS-1 (groups of individuals) to MLS-2 (groups as individuals)66–69.\n\nDespite its controversy-plagued history (see below), the multilevel selection theory has undergone a resurgence of interest in recent years (e.g. 6,12,58,70–72; see 48 for a book-length treatment). This is because it has provided novel perspectives on a variety of issues such as parasitic virulence73, cultural group selection in humans (that is often envisioned as the outcome of warlike confrontation)74,75, or the ‘major transitions’ in evolution48,69.\n\n\nThe controversy\n\nThe controversy surrounding the theories of kin and multilevel selection has a long and turbulent history (detailed, e.g., in 1,24,48,61,76). Until the second half of the last century, many biologists did not clearly distinguish between different levels of selection, and it was often uncritically assumed that group selection would easily prevail over individual selection (e.g. 77) or that individual selection alone would foster adaptations ‘for the good of the group’ (e.g. 78). It was the rebuttal of these ‘naïve’ assumptions (though not of the theoretical plausibility of group-level thinking per se; cf. 6) that widely led to the rejection of group selection as a significant evolutionary force79. Notably, kin selection theory—along with other theoretical frameworks such as evolutionary game theory80 and selfish gene theory37,81—was initially developed as an alternative to group selection (e.g. 11,15), which likely contributed to an increasing polarization in disfavor of arguments based on group-level thinking (e.g. 37).\n\nHowever, the demise of group selection was only temporary. Subsequent studies dropped the ‘naïve’ assumption of the unconditional superiority of group selection and instead acknowledged that selection within groups often undermines selection among groups (e.g. 49,82–84). Building on this premise, trait-group models suggested that group selection can drive evolutionary change even when opposed by within-group selection and that a periodical blending of groups (e.g. in a common mating pool) can prevent the seemingly inevitable fixation of selfish types within groups49,84. Moreover, empirical studies demonstrated that experimentally applied group selection can drive evolutionary change (e.g. 85–87; reviewed in 59,88) and argued that early models had restricted the applicability of group selection by deploying unrealistic assumptions, such as the notion that group and individual selection are always diametrically opposed (reviewed in 6,51). Interestingly, Hamilton himself showed that multilevel selection was formally equivalent with his theory of inclusive fitness (31, see next section), suggesting that the two theories simply outline different perspectives on the same natural processes. In some minds, this realization closed the debate, as the choice between the two theories seemed to have become a mere matter of personal taste. But far from it, the relationship between kin and multilevel selection remained controversial.\n\nOver the last four decades, the group selection controversy has lost little of its initial momentum and continues to polarize opinions fueled by semantic debates (reviewed in 12,65,70) and, ultimately, the different implications the two theories seem to have for the evolution and self-perception of our own species89–91 (see also 92 and associated responses). Accordingly, some biologists contest the usefulness of multilevel selection in the study of social evolution in general12,65, whereas others call for a reframing of the theoretical foundations of sociobiology from a multilevel perspective6. Nevertheless, the focus of the controversy has shifted away from the question of whether group selection occurs at all and now mainly revolves around (the consequences of) its presumed formal equivalence with kin selection theory and the question of whether group selection can lead to (group) adaptation. These are the two topics we will discuss below.\n\nMost biologists consider kin and multilevel selection formally equivalent (e.g. 24,40,61,93,94), but this view is not universally accepted and the number of dissenting voices has recently grown (e.g. 95–102). What, then, is the basis for the formal equivalency of the two approaches, and why is it still controversial?\n\nOn a practical level, the compatibility of kin and multilevel selection relies on the fact that both theories require positive assortment of (genetically) similar individuals for cooperative behaviors to evolve (31,103,104; see 105–108 for examples). From a multilevel perspective, positive assortment increases the scope for between-group selection, as it will make groups internally more homogeneous and thus reduce the potential for within-group selection (Figure 1)48. From a kin selection perspective, positive assortment ensures that costly social behaviors such as altruism are preferentially directed toward individuals that show the behavior themselves31. This directionality is crucial: altruistic traits are selectively disadvantageous even when directed at otherwise (genetically) very similar non-altruists (Figure 1) and thus can evolve only if altruists sufficiently often interact with other altruists, thereby increasing their average inclusive fitness over that of non-altruists31. In practical terms, the compatibility of kin and multilevel selection hence conveys that individuals expressing social behaviors (such as altruism) have a higher inclusive fitness than selfish individuals, whenever selection between groups is stronger than selection within them, and vice versa109.\n\nOn a theoretical level, the compatibility of kin and multilevel selection is conventionally understood to predicate that group selection models can always be recast in terms of inclusive fitness39,93. This formal equivalency is usually inferred by using an equation developed by George C. Price110,111 that expresses the intergenerational, population-level response to natural selection in a heritable trait as the covariance, taken over all individuals within the population, between an individual’s trait and its fitness (here measured as its fecundity; e.g. 31,40,94). The Price equation allows partitioning the evolutionary change into its direct and indirect components and can be used to derive Hamilton’s rule (the kin selection approach;18,22,34,36). However, it also lends itself to partition the evolutionary change into effects at the individual and group levels22,31,40,48,94,111. Hence, kin and multilevel selection are formally equivalent when formulated as alternative decompositions of the Price equation, as both approaches make it possible to correctly compute the total evolutionary change. The approaches differ merely in how this change is partitioned and thus offer different, potentially complementary ways of viewing evolution in structured populations31,40,94,112. Two points, however, deserve a closer examination: firstly, the above-described decomposition of multilevel selection applies only to scenarios of the MLS-1 type (as group fitness is defined as average individual-level fitness of group members;48). Secondly, the multilevel partitioning requires individuals to be nested in non-overlapping groups; the kin selection approach comes with no such requirement and thus is arguably more general within the Price framework24,31,48.\n\nAlbeit most commonly used in theoretical studies, the Price framework is not the only approach to study multilevel selection. An alternative approach (that is often adopted in empirical studies; e.g. 55,113,114) is offered by contextual analysis48,50,115. Like the Price equation, contextual analysis partitions the change due to natural selection (in MLS-1 scenarios;48) into individual and group effects; but unlike the Price equation, it detects group selection only if group effects on fitness remain even after controlling for individual effects. Contextual analysis thus accommodates a classic criticism against the multilevel partition of the Price equation, which can detect a component of between-group selection even in non-social contexts (i.e. when the evolution of the population can be predicted without taking group structure into account), and hence might not always accurately reflect the true causal effect of group selection48,115. However, contextual analysis has problems of its own, as it detects group selection when there is no variation in fitness among groups (e.g. because they all have the same productivity), but individual fitness depends on their ranking within the group (soft selection;48,50,115; but cf. 59). Owing to these problems, it is still controversial which approach is better suited to study multilevel selection (e.g. 48,58–60,116,117; see also 99,118,119). Note however that contextual analysis is formally very similar to modern kin selection models that are based on neighbor-modulated fitness. This supports the conjecture of an equivalency of kin and multilevel selection, as the two approaches seem inter-translatable even when multilevel selection is studied by using contextual analysis rather than the Price equation1,30.\n\nThough suggested by both the Price equation and contextual analysis, the formal equivalency of kin and multilevel selection remains controversial (e.g. 95–99). Most critics seem to reject (aspects of) the generalization of kin selection theory and instead contrast specific, narrowly defined formulations of kin selection with more general approaches to multilevel selection (e.g. 96,99,100,102). For example, Wilson and Hölldobler100 rejected the broad definition of relatedness, arguing that it leads to a departure from the earlier and heuristically very useful narrow definition of kin selection. As a consequence, it can be argued that kin selection is only a special case of multilevel selection because relatedness (i.e. genetic similarity) can occur without strict kinship and hence evolution can occur by group selection in the absence of selection among narrow-sense kin (for instance, if group selection acts on green beards)1. Similarly, Van Veelen and colleagues96,99 (see also 95,97) rejected attempts to generalize kin selection and then contrasted multilevel selection with a specific (rather than a general) version of kin selection in which the cost-benefit parameters of Hamilton’s rule denoted fitness payoffs (rather than partial regression coefficients). They showed that this kin selection approach can lead to incorrect predictions if the payoffs are non-additive (see also 120) and hence concluded that multilevel and kin selection are not equivalent. However, they compared a specific formulation of kin selection with their general formulation of multilevel selection and hence arguably could not refute assertions of the equivalency of the two theories that are based on general formulations of kin selection (cf. 17).\n\nInterestingly, these rejections of the general formulation of kin selection99,121 (see also 122,123) relate to a more extensive debate that was initiated by a high-profile charge of Nowak, Tarnita, and Wilson against the value of inclusive fitness theory in explaining the evolution of eusociality101. This partly philosophical debate revolves around the question of whether the cost-benefit parameters in general formulations of Hamilton’s rule allow a causal interpretation at all. As these parameters denote partial regression coefficients, they can depend on relatedness124 and population gene frequency, which can, for instance, lead to the counterintuitive result that a social behavior satisfies Hamilton’s rule at a low, but not at a high, frequency13. Whereas critics consequently deny the general formulations of Hamilton’s rule any explanatory power and claim that they cannot accurately describe the evolutionary dynamics of any given system (e.g. 13,101,121,125), others argue that they serve as a unifying principle that provides a super-ordinated framework for interpreting the results of otherwise disparate models in a general terminology (e.g. 18,39). Overall, this debate reveals that the formal equivalency of kin and multilevel selection (somewhat obviously) holds only if equally general formulations of the two theories are pitted against each other and that the issue at the heart of the debate really is the question of whether such general formulations make sense from a heuristic perspective (e.g. 13,18,19,101,125–128; see 17,41 for in-depth reviews). Indeed, the question of whether and when general versions of Hamilton’s rule (and thus kin selection theory) provide a better/worse causal (rather than statistical) representation of the evolutionary process than the corresponding general approaches to multilevel selection (see also Conclusions section) might provide a fruitful avenue for future discussions.\n\nAnother line of reasoning against the equivalency of kin and multilevel selection suggests that even generalized formulations of kin selection cannot account for the long-term effects of events on the group level57,95,98. Group-level events such as fission, fusion, or extinction often occur asynchronously, and Simon and colleagues57 recently suggested that group selection should consequently be thought of (and analyzed) as an asynchronous, continuous-time process that is shaped by the combined, long-term effects of such group-level events. On this basis, they argued that although kin and multilevel selection are often equivalent when only one time interval between two group-level events is analyzed, they would almost never be equivalent in a dynamical setting because kin selection approaches could not account for the asynchronous nature of the group-level events57,98 (see also 129). Interestingly, they also suggest that the long-standing disagreement over the equivalency of kin and multilevel selection is based on oversimplified models of multilevel population dynamics and an inappropriate definition (via the Price equation) of group selection57,98: in its usual form, the Price equation traces the evolutionary change only over short periods (see above), assumes that all relevant processes such as reproduction or mass dispersion occur at a discrete set of time points, and is restricted to MLS-1 scenarios in which group-level events (other than mass dispersion) do not feature explicitly57,98. Thus, the Price equation (and contextual analysis) might be insufficient to capture all relevant aspects of the selection among groups.\n\nOverall, the last word on the equivalency of kin and multilevel selection has surely not been spoken, as the partly philosophical character of the debate prevents it from being settled by theoretical or empirical results alone (cf.17). However, even if it would turn out that the equivalency is untenable in some situations, kin and multilevel selection will surely continue to occupy largely overlapping domains, leaving evolutionary biologists with both the blessing and the curse of the existence of multiple theoretical frameworks to study social behavior.\n\nOne fundamental issue that triggered the initial rejection of group selection was the (then naïvely alleged) claim that it can foster group adaptation (i.e. promote the evolution of traits ‘for the good of the group’)6. Although an evolutionary response to group selection has by now been demonstrated in a variety of laboratory and field studies (e.g. 85,130–132), the claim that it can foster group adaptation (or any adaptations at all) remains highly controversial12,65.\n\nRecently, Pruitt and Goodnight56 reported that natural colonies of the social spider Anelosimus studiosus are characterized by a site-specific mixture of ‘docile’ and ‘aggressive’ individuals and showed that experimentally constructed colonies with compositions mimicking the naturally occurring mixtures survived in the field but that colonies with deviating compositions perished. Experimental colonies with a perturbed composition that had survived at a ‘foreign’ site had shifted their composition toward a mixture that would have been optimal at their native site rather than toward the locally optimal mixture. Considering these results, Pruitt and Goodnight suggested that the composition of colonies differs between sites because of site-specific group selection and—as it is optimized to promote long-term colony survival at the native site—constitutes a group adaptation56. This latter conclusion, however, did not go unchallenged133–135. Grinsted and colleagues134 criticized that individual-level selection was not ruled out as an alternative explanation of Pruitt and Goodnight’s results, and Smallegange and Egas133 indeed developed an environmental threshold model to explain Pruitt and Goodnight’s observations at the individual rather than the group level. Finally, Gardner135 argued that colony composition is unlikely to maximize colony fitness and thus rejected the claim that the site specificity of colony composition constitutes a group adaptation.\n\nDo the results of Pruitt and Goodnight56 thus provide no evidence for a group-level adaptation after all? The answer depends on the definition of ‘group adaptation’. In a kin selection framework, adaptations are regarded as occurring at the level of the individual organism and to maximize an individual’s inclusive fitness19,135,136. By analogy, group adaptation is thus understood as a process that is driven by between-group selection and optimizes phenotypes for the purpose of group fitness maximization135,136 (see also 137). This optimization process, however, is typically compromised by within-group selection because of conflicts among group members and thus will be favored by natural selection only if these conflicts are either absent (as for instance in clonal groups) or completely suppressed136 (but cf. 138,139), for example, through mechanisms such as fair meiosis140,141 or worker policing140,142. Accordingly, group adaptations are expected to occur only rarely in nature, where their demonstration would require showing that within-group conflict is absent and group fitness is maximized135,136. Pruitt and Goodnight56 did not assess within-colony conflict and thus arguably provide no conclusive evidence for group adaptation according to the above definition—a view that is embraced by all critics of their interpretation133–135.\n\nHowever, a different approach to group (and individual) adaptation is conventionally taken in a multilevel selection framework (e.g. 109,143,144). In accordance with the kin selection framework, a process would be defined as group adaptation if the trait frequency evolves toward (or has settled down at) the group optimum (i.e. the trait frequency that is predicted to evolve when only between-group selection is at work). Likewise, a process would be defined as individual adaptation in both frameworks if the trait frequency is driven by within-group selection only and hence evolves toward (or has settled down at) the individual optimum. However, differences between the kin and multilevel selection frameworks emerge if within- and between-group selection are aligned or if the metapopulation evolves toward (or has settled down at) a compromise (i.e. an intermediate trait frequency)61,109 (see also 1). In these situations, the (outcome of the) process would be called an individual adaptation in the kin selection framework, where adaptations are generally considered to occur at the level of the individual irrespectively of the strength of between-group selection136,145. In contrast, such compromises are often considered group adaptations in a multilevel selection framework, especially if they are (predominantly) driven by between-group selection109,144. Accordingly, while mechanisms of conflict suppression such as policing and punishment are a prerequisite of group adaptation in the kin selection framework, they are often considered group adaptations themselves in a multilevel selection framework6,84. In a reply to their critics, Pruitt and Goodnight adhere to this latter view and argue that the group-level trait ‘colony composition’ is shaped by site-specific group selection and hence constitutes a group adaptation143.\n\nThis recent debate about the implications of Pruitt and Goodnight’s findings56 sheds light on a clear distinction between the kin and multilevel selection frameworks. The kin selection approach typically grants the individual priority as an evolutionary agent because it appears as an adaptive unit and consequently allows a clear-cut distinction between individual and group adaptations even if selection acts on both the within- and between-group levels136,145 (but cf. 1,61,109). In contrast, the multilevel selection approach allows such a clear distinction only in special cases (namely if selection acts only on one level) but places more emphasis on the fact that the realized frequency of a social trait is usually a compromise of different selection pressures109,143. Moreover, the multilevel selection approach allows for situations in which selection pressures on the individual and group levels are (at least to some extent) aligned. This might be the case where collective traits (e.g. the superstructure of waterproof rafts built by fire ants through self-assembly146) simultaneously promote the survival of the group and directly benefit the individuals within it. We suggest that both approaches may provide important insights into our understanding of social evolution and that, instead of focusing on their (semantic) differences, a more fruitful approach to the adaptiveness of social groups might be to ask how well adapted a particular social group is relative to the (theoretical) ideal of a conflict-free group1 (see also 136,147). Moreover, it is important to note that although the kin selection approach to group adaptation is more restrictive than the multilevel selection approach, both in principle allow for group adaptation. Thus far, the formal demonstration of group adaptation (i.e. a group-optimal and conflict-free outcome) according to Gardner and Grafen’s kin selection-based definition136 is still pending. However, elaborate group-level traits such as the dance-language of honey bees148 are good candidates that might live up to the definition of group adaptation within both frameworks.\n\n\nConclusions\n\nKin and multilevel selection are two key concepts of modern sociobiology that provide different perspectives on the evolution of social behaviors. Unfortunately, these approaches are often pitted against each other in a seemingly endless (and largely semantic) debate that arguably impedes scientific progress91 and prevents the benefits of the different perspectives from being harnessed.\n\nMost biologists prefer kin selection over multilevel selection approaches as a matter of habit or personal taste12,65, and this preference seems partly justified as kin selection approaches have received more theoretical attention (and are hence highly versatile)20,23 and have been put to work in more empirical applications17,46,149 (but note that the widespread acceptance of their formal equivalency implicates that empirical evidence for one theory cannot be used as evidence against the other). However, we believe that a bipartisan view on the kin and multilevel selection theories might ultimately prove more fruitful. After all, there might be situations in which one approach provides a more accurate representation of the causal structure of social interactions despite their (presumed) equivalency as statistical decompositions of evolutionary change17,150. For example, it might be more causally apt to describe the selection pressures on a segregation distorter allele that has negative effects on the fitness of its bearer in terms of multilevel selection (i.e. as opposing selection pressures at the gene and individual levels). Conversely, it might be more causally apt to describe the selection on cooperative behavior in pairwise interactions between related individuals in terms of kin selection, especially if those pairs are ephemeral and form only for the duration of the social interactions17. More generally, kin selection might provide a more accurate representation of the causal structure of social interactions than multilevel selection where fitness pertains to individuals in the first instance (and group fitness is a simple function of the fitness of its constituent individuals; see also 22,48), whereas the opposite might be true where fitness pertains to the whole group in the first instance (and individual fitness is determined by group fitness)150. Such considerations of causal aptness might help to explain why multilevel selection was readily accepted for the study of major transitions48,69 but only slowly establishes itself in the field of behavioral ecology, in which social interactions are often studied in ephemeral ‘groups’ of genealogical kin42. It is noteworthy that considerations of causal aptness seem less clear in the case of eusocial systems (such as ants, termites, and some species of bees and wasps; cf. 48) and that exactly these systems take center stage in the controversy surrounding the kin and multilevel selection theories (e.g. 100,101,126,127,151).\n\nInterestingly, kin and multilevel selection approaches might ultimately prove to be very useful exactly when applied to the same system. Kin selection analyses often follow an optimality (‘adaptationist’) approach and accordingly try to identify the phenotype(s) with the highest overall fitness to extrapolate where a population will eventually stabilize18,30. The strength of kin selection (as a driver of evolutionary change over the course of one or multiple generations), however, is rarely reported30. In contrast, multilevel selection approaches often follow an ‘evolutionary change’ approach and examine how a population will change its current configuration (for example, depending on the strength of within- and between-group selection or in response to an applied selection pressure)30,59. The optimal phenotype, however, is typically not identified in multilevel selection studies30. The two perspectives opened up by the kin and multilevel selection approach, respectively, seem to be highly complementary30,59. Experimental studies already began to harness the translatability of the two approaches (e.g. 107,108,152,153). However, we still eagerly await studies that make use of the full potential of their complementarity by combining both the ‘evolutionary change’ and ‘adaptationist’ perspective and the methods that come along with them. We believe that such studies would go a long way toward gaining a deeper understanding of the processes that ultimately drive the evolution of social behaviors in structured populations.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis study was supported by the German Science Foundation (DFG; ME4179/1-1 to JM).\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 Susanne Foitzik, James A.R. Marshall, Heikki Helanterä, Jonathan Pruitt, and Rolf Kümmerli for their helpful comments on an earlier version of this manuscript.\n\n\nReferences\n\nFoster KR: A defense of sociobiology. Cold Spring Harb Symp Quant Biol. 2009; 74: 403–18. PubMed Abstract | Publisher Full Text\n\nWilson EO: Sociobiology: The new synthesis. Harvard University Press, 1975. Reference Source\n\nSachs JL, Mueller UG, Wilcox TP, et al.: The evolution of cooperation. Q Rev Biol. 2004; 79(2): 135–60. 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}
|
[
{
"id": "13637",
"date": "28 Apr 2016",
"name": "James Marshall",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13636",
"date": "28 Apr 2016",
"name": "Rolf Kümmerli",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13635",
"date": "28 Apr 2016",
"name": "Jonathan Pruitt",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "13634",
"date": "28 Apr 2016",
"name": "Heikki Helanterä",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-776
|
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